(Circulation. 1996;93:1447-1458.)
© 1996 American Heart Association, Inc.
Articles |
Intrinsic Myocyte Dysfunction and Tyrosine Kinase Pathway Activation Underlie the Impaired Wall Thickening of Adjacent Regions During Postinfarct Left Ventricular Remodeling
From the Cardiology Division, Department of Medicine, and the Division of Magnetic Resonance Imaging, Department of Radiology of the Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, Md.
Correspondence to João A.C. Lima, MD, Cardiology, Blalock 569, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287.
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Abstract |
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Background Left ventricular remodeling after infarction is accompanied by dysfunction of regions adjacent to the infarct. We hypothesized that myocyte contractile abnormalities and elongation greater than in remote regions underlie adjacent-region dysfunction in the remodeled ventricle. The activation of the tyrosine kinase pathway, which mediates in vitro hypertrophy by stretch and/or angiotensin, was also assessed in myocytes separately isolated from adjacent and remote regions.
Methods and Results ECG-gated magnetic resonance imaging short-axis images were acquired 2 weeks after coronary ligation in rats. After the rats were killed, myocytes were isolated from animals with large (n=7) and small (n=7) infarcts and from 4 sham-operated controls. Regional wall thickening was correlated with local myocyte function and morphology. Cytochemistry for tyrosine-phosphorylated proteins was performed in myocytes from the same regions. Remodeled ventricles were dilated relative to controls by 93.7%, and wall thickening in adjacent regions was less than in remote regions (27.8±6.11% versus 54.0±10.1%, P<.01). In large infarcts, cell extent and velocity of shortening were reduced in adjacent cells versus controls by 47% and 44%, respectively (P<.05). Myocyte shortening was reduced in adjacent versus remote regions (P<.06), and cell dysfunction correlated with impaired wall thickening (r=.72, P<.05). Myocytes in adjacent regions were longer than in remote regions (150.3±1.89 versus 143.1±1.76 µm, P<.05) and also showed 88% more membrane-related phosphotyrosine clusters (P<.05).
Conclusions After infarction, impaired wall thickening in adjacent regions is accompanied by greater myocyte dysfunction and elongation than in remote regions. These abnormalities are associated with regional differences in the tyrosine kinase pathway activation, indicating a potential intracellular mechanism for postinfarct myocardial remodeling.
Key Words: myocardial infarction • remodeling • magnetic resonance imaging • heart failure • tyrosine kinase
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The global alterations of left ventricular architecture that characterize left ventricular postinfarct remodeling consist of ventricular dilatation1 and distortion2 3 accompanied by eccentric hypertrophy,4 5 which occur over the course of weeks to months.1 2 3 4 5 Typical histological features of postinfarct ventricular remodeling include side-to-side myocyte slippage6 and myocyte hypertrophy,7 8 9 initiated early after myocardial infarction to accommodate the progressive increase in end-diastolic volume that characterizes the process. Although this process is believed to constitute a compensatory response to acute myocardial loss, it is associated with progressive chamber dysfunction and an increased incidence of sudden death, congestive heart failure, and thromboembolism.10 11 12 13 14 15 16
Serial follow-up of left ventricular dimensions and regional function after myocardial infarction reveals an early17 18 and persistent5 difference in segmental performance between noninfarcted adjacent and remote regions. Regional histological differences in the extent of myocyte hypertrophy have also been found in animal models of left ventricular remodeling.7 8 These observations suggest that regional differences in mechanical load3 5 17 19 or humoral factors acting in an autocrine or paracrine fashion20 21 22 may play a major role in the progression of the postinfarct remodeled heart toward end-stage pump failure. They support the concept that postinfarct remodeling is in essence a regional process that progresses from adjacent noninfarcted regions to involve the entire left ventricle as more and more contractile units become exposed to the damaging effects of mechanical overload and/or humoral overstimulation.
Mechanical overload, such as that seen in postinfarct remodeling, results in left ventricular hypertrophy,23 which can also be induced by exposure to several growth factors and vasoactive substances, including angiotensin II.20 21 22 24 25 Moreover, evidence from experiments performed in isolated myocytes indicates that both humoral and mechanical stimulation converge to activate tyrosine kinase enzymes (or inhibit tyrosine phosphatases), initiating a cascade of secondary messages that lead to cell hypertrophy.26 Thus, the ubiquitous intracellular tyrosine kinase pathways are involved not only in the mitogenic response to growth factors in cell lines capable of division but also in the cardiomyocyte hypertrophic response to stretch27 and humoral stimulation.20 21 22 24 25 However, despite the growing availability of data supporting the central role played by the tyrosine kinase pathway in intracellular protein synthesis associated with hypertrophy, evidence that this pathway is activated during conditions eliciting in vivo hypertrophy in adult cardiomyocytes is still lacking.
We hypothesized that regional differences in segmental performance detected during the postinfarct remodeling process reflect regional morphological and functional differences in the corresponding myocyte populations. In addition, we hypothesized that if the tyrosine kinase signaling pathway plays a role in the compensatory hypertrophic response to myocardial loss by infarction, regional differences in the activation of this system would parallel regional myocyte morphological and functional differences. We found differences in contractile function and myocyte morphology between cells isolated from adjacent and remote noninfarcted regions 2 weeks after myocardial infarction. These cellular alterations were associated with (1) increased protein tyrosine phosphorylation of membrane-associated proteins in myocytes isolated from adjacent regions and (2) locally impaired segmental wall thickening demonstrated in vivo by magnetic resonance imaging (MRI).
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Methods |
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Experimental Preparation
A total of 39 male Sprague-Dawley rats weighing 200 to 225 g (Harlan) were used in the study. Each animal was assigned to either of two groups: (1) rats subjected to total left coronary occlusion or (2) sham-operated controls. Under anesthesia with inhaled methoxyflurane (administered with a nasal cone), intercostal incision was performed to expose the heart: the pericardium was opened and the left coronary artery ligated. Sham-operated animals underwent a similar procedure except for the ligation of the coronary vessel. Successful ligation of the coronary artery was immediately assessed by evidence of cyanosis of the anterior left ventricular wall and later confirmed by postmortem macroscopic and histological presence of myocardial necrosis. In the group subjected to total left coronary occlusion, 9 of 31 rats (29%) died within 72 hours after surgery, and 1 of 8 sham-operated rats (12%) died immediately after surgery.
The surviving 29 rats (22 infarcted and 7 controls) were killed 2 weeks after surgery. In 18 animals (14 infarcted and 4 controls), the heart was perfused with collagenase to obtain isolated cardiomyocytes; in 9 rats, the hearts (6 infarcted and 3 controls) were fixed for histological studies, and in 2 infarcted hearts a mock isolation protocol was performed as described below.
MRI Protocol
MRI was performed in 12 rats (4 sham-operated and 8
infarcted animals) within the 12 hours preceding euthanasia. Images
were acquired in a 1.5-T whole-body Signa scanner (General
Electric) with a 3-in surface coil. The animals were positioned in the
scanner after sedation with sodium pentobarbital (30 to 50 mg/kg IP),
and continuous ECG monitoring was performed.
In preliminary experiments, we found that the heart rate of the rat
(
300 beats per minute) was too high for the scanner and that the
amplitude of the ECG R wave was often too small, particularly in rats
with myocardial infarction. To overcome these technical problems, we
constructed specialized hardware that intercepted the electrical signal
used by the scanner for cardiac gating and passed only every other
pulse, effectively dividing the actual heart rate by two (eg, an actual
rate of 300 beats per minute was sensed by the scanner as 150 beats per
minute). In addition, we increased the amplitude of the ECG waveform
using the variable gain on a Gould BioTach ECG module (Gould
Instruments) before sending the ECG signal to the scanner. These
technical modifications allowed us to image rats by use of ECG gating
on the GE Signa scanner.
ECG-gated spin-echo images were obtained with the following imaging parameters: TR, 4 RR intervals; TE, 13 ms; field of view, 8 cm with in-plane pixel size of 0.30x0.60 mm (interpolated to 0.30x0.30 mm); and slice thickness, 3 mm. The relative in-plane resolution was similar to that of MRI studies in larger mammals5 or in humans when performed using spin-echo sequences. For instance, in human studies, the typical in-plane pixel size is 1x1.5 mm, interpolated to 1x1 mm; thus, in a short-axis view, the distance between the endocardium and epicardium is covered by about 10 interpolated pixels. In the rat, the left ventricular thickness ranges from roughly 3 to 4 mm, ie, about 10 interpolated pixels in a short-axis view having the resolution reported in this study.
Coronal and sagittal scout views were first obtained to identify the oblique planes of interest in short-axis views of the left ventricle. In each animal, three short-axis planes were acquired to span the left ventricle from base to apex. For each short-axis plane, a protocol of alternated multiple image acquisition was used to allow imaging of multiple phases during the cardiac cycle: 6 to 9 images equally spaced (20 ms) from the triggering R wave were collected for each plane.
The sequential display of the MRI frames in continuous loop facilitated the identification of epicardial and endocardial contours for measurement of left ventricular cavity size and systolic wall thickening. The infarcted region was readily identified by end-diastolic wall thinning in the MRI spin-echo T1-weighted short-axis images5 : the infarct was typically well defined, with sharp borders.
For the assessment of global left ventricular areas and
wall thickness, the short-axis image plane taken at the level with
the maximal circumferential extent of infarcted tissue was selected for
quantitative analysis. The end-systolic (smallest
cavity area) and end-diastolic (obtained 13 ms after
triggering upstroke of the ECG R wave) frames were used for
computer-assisted contour analysis to quantify left
ventricular endocardial cavity area (area outlined by the
endocardium in the short-axis view) and ratio of endocardial cavity
area to myocardial area (the latter defined as the difference between
the areas outlined by the epicardial and endocardial contours in the
short-axis view; Fig 1
). The analysis of
segmental systolic wall thickening from cross-sectional
short-axis images of the left ventricle has been described in
detail.17 28 Briefly, the myocardial area enclosed by the
endocardial and epicardial contours of left ventricular
short-axis images is divided into 16 radial segments, and the wall
thickness for each segment is computed as the ratio of segment area to
the averaged epicardial and endocardial segmental arc lengths.
Systolic wall thickening for each segment was computed as
end-systolic minus end-diastolic wall
thickness divided by the end-diastolic wall thickness.
The adjacent noninfarcted region was defined as the 2 or 3 segments
adjacent to each side of the infarct scar.5 17 The
segments located on the opposite side of the infarct (generally
comprising the interventricular septum) in the most
basal image plane were considered remote, as previously
described.5 In most infarcted hearts (5 of 8), that plane
was not involved by the infarct and therefore was separated from the
scar by at least 1 image plane. Corresponding segments were used for
segmental functional analysis in sham-operated
controls.
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In a subgroup of animals (1 sham-operated, 3 infarcted animals), intraobserver and interobserver variability were also assessed. The same observer (intraobserver variability) and two independent observers (interobserver variability) repeated twice the measurement protocol described above. For each parameter, the variability was quantified as the percent ratio of the difference to the average of the two observations. Intraobserver variability of MRI measurements averaged 2.68% in area measurements and 9.82% in thickening measurement. Interobserver variability was 4.15% and 13.22%, respectively.
Regional Myocyte Isolation and Infarct Size
The animals were killed by administration of sodium
pentobarbital overdose (75 mg/kg) 2 weeks after surgery, and single
cardiac myocytes were isolated according to a modification of a
previously described protocol.29 Briefly, the heart was
excised and retrogradely perfused with a low-Ca2+
collagenase-containing bicarbonate buffer (37°C, pH
7.3). When the heart became soft, the perfusion was stopped and the
heart removed from the cannula. The right ventricular free
wall and the atria were separated from the left ventricle. The left
ventricle was then opened with a cut bordering the anterior aspect of
the interventricular septum along the longitudinal axis
from base to apex (interventricular sulcus) (Fig 2). Macrophotographs (Nikon N2000) of the heart were
taken to measure infarct size. These photographs were later digitized
for computer-assisted planimetry to quantify the relative extent of
the scar as a percentage of the subepicardial left
ventricular surface.
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Small wedges of tissue (volume, 50 mm3) were cut from
the region bordering the infarct scar (adjacent region) and from the
region opposite the infarct scar (remote region). Special care was paid
to completing this part of the procedure as quickly as possible: within
3 minutes from the discontinuation of the perfusion flow, the samples
had been separately minced and the myocytes filtered and resuspended in
the first of a series of bicarbonate-based buffers (in mmol/L: NaCl
123, KCl 5.4, MgSO4 1.2, NaH2PO4
1.2, glucose 5.6, and NaHCO3 20) with increasing
Ca2+ concentrations. Cells were finally suspended in
HEPES-based buffer (in mmol/L: NaCl 137, KCl 5.4,
Ca2+ 1.0, MgSO4 1.2,
NaH2PO4 1.2, glucose 15, and HEPES 20).
In the present study, the above-described method of collagenase perfusion, when performed on control hearts, yielded a suspension containing 70% to 80% of intact rod-shaped myocytes. An average of 60% of intact myocytes were obtained in samples from the remote region and 45% from the adjacent area in infarcted hearts. The different cell viability noted in the two areas, not evident in the sham-operated animals, was already present before cells were reexposed to calcium. The mechanisms responsible for this difference are partially unknown, although similar findings have been reported by other investigators29 and have been attributed to the surgical procedure and to a relative increase in collagen within the extracellular matrix of adjacent noninfarcted regions. Compared with the usual isolation technique performed on the entire left ventricle, this modified protocol yielded a much smaller total number of myocytes because small volumes of myocardium were sampled from the left ventricle to avoid overlap between adjacent and remote noninfarcted regions. For this reason, rod-enrichment procedures (serial centrifugation or centrifugation in Percoll gradients) could not be performed to reduce the contamination of myocyte suspensions because they cause further myocyte loss.
Morphological and Functional Studies of Isolated
Myocytes
Cardiac myocytes (Fig 3) were imaged in an
open perfusion cell bath at room temperature (25°C) with a Nikon
inverted microscope connected to a television camera (Javelin
Electronics); the images were directed to a line-scan electronic
edge-tracker device (Crescent Electronics) whose output was
connected through an analog-to-digital board (National
Instruments AT MIO 16F 5) to a 486 PC (Dell) for
analog-to-digital conversion and measurement of cell
dimensions. During the data acquisition, the cells were superfused with
HEPES-based buffer containing glucose and 1 mmol/L Ca2+.
Cell length and width were recorded in each animal from 25 to 30
electrically unstimulated myocytes from each region (adjacent and
remote). The ratio of cellular length to width was also computed and
included in the analysis. In the control group, cell width and
length were measured in 40 to 45 cells from the left
ventricular free wall and left ventricular
septum (total number of cells, 175 from each region) and compared with
adjacent and remote areas, respectively, from the small- and
large-infarct groups (total number of cells, 200 from each region
for each group).
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Myocyte percent systolic shortening and velocity of cell shortening were measured in 5 to 10 randomly chosen myocytes from each region of each heart (total number of cells for each region: 35 for the large-infarct group, 42 for the small-infarct group, and 20 for the sham-operated controls). Unloaded percent systolic shortening and velocity of cell shortening were measured during stimulation at 1 Hz with rectangular current pulses 5 ms in duration and twice diastolic threshold through platinum field electrodes. To avoid investigator selection bias, the cells were chosen for recording before electrical stimulation was begun. The parameters for each myocyte were averaged from 10 consecutive high-quality beats acquired at steady state after 2 minutes of pacing. Digitized pictures of sampled myocytes isolated from sham-operated and infarcted rats were used to measure the average sarcomere length from the distance encompassing 25 and 50 contiguous sarcomeres. Because the average sarcomere length was >1.85 µm in all cases, the possible inclusion of hypercontracted cells in the sampled population was eliminated.
Histological Assessment of Cell Diameter and
Myocardial Fibrosis
Histological measurements of cell diameter were
performed in an additional subgroup of 9 rats (3 controls and 6 animals
with large infarcts) killed 2 weeks after surgery. After
perfusion-fixation, each heart was serially sliced into
2-mm-thick rings perpendicular to the longitudinal axis from the
base to the apex. From each slice, embedded in glycomethacrylate,
sections were cut at a thickness of 3 µm with a Historange microtome.
The sections were stained with toluidine blue and mounted on glass
slides for microscopic analysis (Fig 4).
Computer-assisted measurements of cross-sectional myocyte
surface area were obtained from cells in the adjacent and remote
noninfarcted regions defined as described above (see "Regional
Myocyte Isolation and Infarct Size"). Adjacent regions were defined
as myocardium within 2 mm of the infarct scar, and remote
as the region opposite the infarct scar. Only cell sections with a
clearly identifiable central nucleus were selected for the
analysis. Myocyte diameter was then calculated under the
assumption that it was equivalent to that of a circle having an area
equal to the measured cross-sectional surface, as previously
reported.7 9
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In 2 additional infarcted rats, a mock isolation protocol was
performed: samples from the different regions of the left ventricle
were removed (adjacent, remote, and scar) and then fixed and stained
for conventional histology. Microscopic analysis of the tissue
isolated from the region visually identified as infarct scar revealed
transmural fibrosis, which was absent in samples taken from the remote
noninfarcted area (septum). Only sparse areas of fibrosis were found in
noninfarcted myocardial tissue adjacent to the infarct scar (Fig 4
). In
the control group, the sham surgical procedure never resulted in the
presence of scar tissue.
Staining of Myocytes for Tyrosine
Phosphorylated Proteins
We used immunocytochemical methods to assess and localize
intracellular tyrosine kinase activity. These methods have been
described in detail in studies from our laboratory30 and
from other groups.31 Briefly, in 3 control rats and 4 with
a large infarct, aliquots of cardiomyocytes from each
region were fixed in formaldehyde (3.7% in PBS) for 1 hour at room
temperature in the dark and permeabilized by a
10-second dip in -20°C acetone. After permeabilization, the
cells were incubated in PBS/1% BSA for 30 minutes at room temperature
in the dark. The cells were then exposed to the first antibody,
anti–phosphotyrosyl residue monoclonal antibody from Upstate
Biology at a concentration of 25 µg/mL in PBS/BSA for 1 hour at room
temperature away from light. After 30 minutes in PBS/BSA, cells were
incubated with the secondary antibody, a goat anti-mouse polyclonal
antibody from Sigma (25 µg/mL in PBS/BSA) labeled with the
fluorescent compound FITC (1 hour at room temperature and away
from light). After incubation with the secondary antibody, cells were
washed rapidly three times in PBS/BSA. The coverslips were finally
incubated overnight in PBS/BSA, then mounted on glass slides (Cytoseal
60 mounting medium). Negative controls were also prepared, with the
primary antibody step in the above-described preparation protocol
omitted.
Only cells incubated with anti-phosphotyrosine antibodies showed the presence of small focal areas of very intense staining: the number of these clusters of tyrosine kinase substrates on rod-shaped myocytes was then averaged in a sample of 20 cells from each region in each animal to indirectly estimate tyrosine kinase/phosphatase enzymatic activity.
This method restricts the evaluation of phosphotyrosine clusters to intact rod-shaped myocytes and prevents data contamination from nonmyocyte cellular fractions and myocytes damaged during the isolation process. Although the number of phosphotyrosine clusters does not quantify tyrosine kinase enzyme content or activity, it reflects the local accumulation of specific enzymatic byproducts and was used primarily to allow relative comparison between two myocyte populations from the same heart.
In addition, confocal laser microscopy was used for imaging of the cell preparations at high magnification (x600). The instrument (Nikon Optiphot-Biorad MRC 600) was equipped with an Ion Laser Technology model 5425 laser head and used a computerized stage for z-axis movements. The excitation wavelength used in the detection of FITC-labeled proteins was 488 nm, and the fluorescence (emission) was measured with a 530-nm filter. Planar images (focusing on planes 1 to 1.5 µm thick) and z-series scans of representative myocytes were acquired (10 parallel sections 1.5 µm thick equally spaced along the axis orthogonal to the slide plane).
Statistical Data Analysis
Repeated measures ANOVA combined with Student-Newman-Keuls
t tests were used to examine the statistical significance of
regional differences in myocyte morphology and function as well as
segmental function by MRI; differences were considered statistically
significant if P<.05. The Student-Newman-Keuls test was
applied whenever the F test indicated the presence of significant
differences. ANOVA was used to compare parameters from
different groups of animals (large infarcts, small infarcts, and
controls). Regression analyses were used to examine the
relation between parameters of cell morphology and infarct
size and between cellular and segmental dysfunction. Results are
presented as mean±SEM.
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Results |
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Left Ventricular Structure and Regional Function
Left ventricular cavity size, indexed by the end-diastolic endocardial area, was greater in infarcted animals (0.62±0.05 cm2, n=8) than in sham-operated animals (0.32±0.04 cm2, n=4; P<.005; Fig 5
). In addition, the ratio of
endocardial area to myocardial wall area was also increased in
infarcted versus sham-operated animals (1.51±0.13 versus
0.83±0.15; P<.01; Fig 5), reflecting the magnitude of
ventricular remodeling 2 weeks after myocardial infarction.
The average infarct size in the animals imaged by MRI was 21.6±3.4%.
In these animals, the scar was easily identified on the basis of
end-diastolic wall thinning.5
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Percent systolic wall thickening was less in the segments
adjacent to the scar than in the remote segments (27.8±6.11% versus
54.0±10.1%; P<.01; Fig 6). In the
infarcted region, systolic wall thickening was substituted by
wall thinning (average percent systolic thickening,
-8.5±6.18%; P<.05 compared with adjacent and
remote). No regional differences in left ventricular wall
thickening were found in the sham-operated rats (59.7±1.6%,
59.6±9.05%, and 59.8±4.8% in the septum and anterior and posterior
free walls, respectively; P=NS; Fig 6).
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Isolated Myocyte Function
The isolated myocyte data analysis was performed in
animals with large infarcts (infarct size, >20%; average infarct
size, 24.8±1.4%; n=7; Table 1) and small infarcts
(infarct size, <20%; average infarct size, 10.1±1.7%; n=7). Myocyte
percent systolic shortening in the adjacent region of hearts
with large infarcts was depressed with respect to controls (6.6±0.67%
versus 9.6±0.67%; P<.05; Table 2, Fig 7). In the same group, myocyte shortening was also lower
in adjacent than in remote regions (6.6±0.67% versus 8.7±0.90%;
P<.06). No significant differences were found when percent
systolic shortening in cells isolated from the remote region of
the large-infarct group was compared with myocyte function from the
corresponding regions of control animals.
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) or rats with
small infarct (
) or large infarct (
). Error bars
represent SEM. *P<.05; +P=.06. Myocytes
isolated from the adjacent noninfarcted region of hearts with large
infarcts show decreased extent of shortening and velocity of shortening
with respect to cells isolated from a corresponding region in controls.
Extent of shortening is also reduced in the adjacent region with
respect to the remote region within the large-infarct group. MI
indicates myocardial infarction.
Similar regional differences in myocyte performance were
observed for velocity of shortening. However, differences in velocity
of shortening were statistically significant only when cells from
adjacent noninfarcted regions of animals with large infarcts were
compared with cells from corresponding regions of sham-operated
controls (P<.05, Fig 7). Moreover, in animals with large
infarcts, the MRI wall thickening in adjacent segments relative to
remote segments correlated directly with the percent extent of
shortening (y=0.42+12.48x;
r=.69) and velocity of shortening
(y=4.87+57.22x; r=.72;
P<.05 for both) obtained from myocytes isolated from those
regions.
The mechanical performance of isolated myocytes in the
small-infarct group did not differ from controls either in the
adjacent or in the remote regions. In the control group, mechanical
performance was homogeneous, as shown by the nearly
identical values of the measured functional parameters in
corresponding regions of the left ventricle (Fig 7).
Myocyte Morphology
Myocyte length was increased by 18% (P<.05) in cells
isolated from the remote region of animals with large infarcts in
comparison with cells isolated from the corresponding regions (septum)
in the control group (Table 2). Similarly, a 17% (P<.05)
increase in the ratio of cell length to width was present in
animals with large infarcts. However, a more pronounced pattern of
morphological changes was present in the adjacent region: average
myocyte length and length-to-width ratio were greater (23% and
26%, respectively; P<.05 for both) than in cells isolated
from the corresponding regions (left ventricular free wall)
of controls. Both cell length and length-to-width ratio were
greater in the adjacent than in the remote region in animals with large
infarcts (5% and 10%, respectively; P<.05 for both).
Myocyte length and length-to-width ratio were also
significantly increased in the large-infarct group with respect to
the small-infarct group (Table 2). However, differences in cell
length and length-to-width ratio between adjacent and remote
regions in animals with small infarcts were not statistically
significant. In addition, cell dimensions in animals with small
infarcts did not differ from those in the sham-operated group
(Table 2).
The average cell length in each animal directly correlated with infarct
size in the adjacent region
(y=118.33+1.271x;
r=.78) and in the remote region
(y=114.88+1.092x;
r=.81; P<.05 for both). By contrast, no
differences were found in myocyte width measured directly from isolated
myocytes in animals with large infarcts, those with small infarcts, or
sham-operated controls (Table 2). Measurements of cell diameter
from histological preparations yielded similar results,
since cell diameter did not differ between hearts with large infarcts
and controls in either the adjacent zone (23.0±0.65 versus 21.2±0.69
µm; P=NS) or the remote zone (23.6±0.66 versus 22.2±0.60
µm; P=NS).
The average sarcomere length did not differ between controls and hearts
with large infarcts (1.91±0.02 versus 1.95±0.04 µm): these results
exclude the presence of sarcomere overstretch or damage secondary to
ventricular dilatation during the remodeling process as
potential mechanisms of cell elongation. They also suggest that cell
elongation during postinfarct remodeling results from the "in
series" addition of new sarcomere units (Fig 3).
Intracellular Tyrosine Protein
Phosphorylation
Immunohistochemistry for tyrosine kinase substrates in single
myocytes showed small focal areas of very intense staining on a
background of diffuse cytoplasmic staining in myocytes from normal and
abnormal hearts (Fig 8). However, clearly distinct focal
areas of intense staining were more frequent in the myocytes from the
adjacent region of hearts with large infarcts compared with those from
the remote region of the same hearts (23.5±3.40 versus 12.5±1.55;
P<.05) or with those from control hearts. By contrast, no
regional differences were present in the control animals
(12.3±0.51 and 15.0±1.5; P=NS), where the average count
was similar to that in the remote regions of animals with large
infarcts.
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Confocal microscopy on thin myocyte sections showed that these small
areas (0.5 to 1 µm in diameter) of focal tyrosine kinase substrate
accumulation were localized predominantly along the inner aspect of the
cell membrane (Fig 9). In addition, confocal microscopy
showed the preferential localization of such clusters in regions where
myocytes branched to connect with each other. These findings support
the concept that tyrosine kinase activation (or tyrosine phosphatase
inhibition) near the plasma membrane of cardiomyocytes is
greater in the adjacent than in the remote noninfarcted regions after
transmural myocardial infarction.
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Discussion |
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This study documents concurrent myocyte elongation and dysfunction in regions of impaired systolic wall thickening located adjacent to transmural experimental infarcts. It is the first study to relate regional left ventricular dysfunction in the intact animal with a well-developed infarct to myocyte dysfunction and hypertrophy in cells later isolated from similar regions of the same hearts. Furthermore, this study examines a possible mechanistic explanation for myocardial remodeling, because we document an increased concentration of membrane-associated proteins phosphorylated on tyrosine in morphologically altered myocytes isolated from adjacent noninfarcted regions.
Regional Myocyte Dysfunction During Ventricular
Remodeling
Regional dysfunction in adjacent nonischemic or
noninfarcted regions has been well documented after coronary
occlusion in humans32 and in several experimental
models.17 18 33 In this study, alterations of myocyte
function in the adjacent region paralleled regional myocyte
elongation and correlated well with in vivo wall thickening impairment
measured by MRI. These cellular alterations are particularly important
in light of evidence implicating adjacent regions as the point of
origin for malignant ventricular arrhythmias in the
postinfarct remodeled ventricle.34 They also correlate
well with previous work demonstrating alterations of
metabolism,35 36 adrenergic
innervation,37 and extracellular matrix38 39
in myocardium adjacent to the infarct border. We believe
that a heterogeneous load distribution in the infarcted
ventricle, resulting from complex changes in chamber shape and
stress,3 5 17 19 40 41 modulates regional myocardial
remodeling, leading to greater elongation in noninfarcted
myocardium adjacent to the infarct border. Such mechanical
overload could also underlie an earlier transition from compensatory
myocyte elongation to elongation and dysfunction in adjacent
noninfarcted regions. In agreement with this hypothesis, we document a
good correlation between impaired segmental thickening and regional
cellular dysfunction in animals with large transmural
infarcts.
Conversely, it can also be hypothesized that molecular changes in cardiomyocytes located closer to the infarct border could be caused by autocrine or paracrine mechanisms leading to myocyte elongation, ventricular shape changes, and segmental dysfunction, as documented in our study. Moreover, the possibility that decreased myocyte velocity of shortening might represent an adaptation to increased mechanical load cannot be excluded entirely from our data. Previous studies have documented a shift to the V3 myosin heavy-chain isoform in the remodeled postinfarct rat myocardium.42 This shift to a fetal pattern, which exhibits a slower contraction, might contribute to but does not totally explain our findings, since adjacent-region dysfunction during postinfarct remodeling has been shown in the canine17 18 33 and ovine experimental models of infarction as well as in humans,3 32 who are known not to undergo myosin heavy-chain isoform shifts in response to hypertrophy.5
The association between depressed ventricular function and reduced mechanical performance of isolated myocytes has been demonstrated in different models of volume overload–induced heart failure.43 44 In the postinfarct remodeled ventricle, myocyte dysfunction has been demonstrated as early as 1 week after coronary occlusion,43 at a time when chamber dysfunction has been well documented in the same experimental model.4 However, previous studies have not investigated regional differences in myocyte performance during left ventricular postinfarct remodeling and have not examined the possibility that cells in the adjacent noninfarcted regions could progress toward dysfunction and failure at a greater rate than other myocyte populations within the noninfarcted regions of the left ventricle.
Intracellular Mechanisms of Myocardial Remodeling
The accumulation of clusters of phosphotyrosine residues in
myocytes isolated from adjacent noninfarcted regions suggests the
involvement of the tyrosine kinase pathway in the cellular alterations
that characterize myocardial remodeling after infarction. Recently,
increasing interest has been focused on the signal transduction
pathways linking external mechanical stimuli to intracellular gene
regulation and myocyte hypertrophy. In addition to the
capability of mechanical stretch and humoral factors to independently
initiate such a hypertrophic response in vitro, it has also been shown
that transduction of mechanical stimuli can activate paracrine
signals to coordinate and potentiate similar responses in neighboring
cells. For example, in cultured cardiac myocytes, mechanical stretch
elicits angiotensin II secretion: this agonist, upon
binding to its AT1 receptor, mediates stretch-induced
hypertrophy through a protein kinase cascade that
critically depends on tyrosine phosphorylation of
specific substrates.45 These new observations strengthen
the concept that angiotensin II plays a central role in the
remodeling process and may improve our understanding of the beneficial
effect of angiotensin-converting enzyme inhibition in
postinfarct remodeling, demonstrated in both
experimental12 and clinical11 15
studies.
The association between cardiomyocyte dysfunction and increased protein tyrosine phosphorylation in the adjacent noninfarcted region of the remodeled left ventricle supports a potential role of the tyrosine kinase signaling system in postinfarct myocardial remodeling. By integration of the information gathered from MRI, isolated myocyte studies, and intracellular biochemical analysis, adjacent regions are characterized by (1) depressed in vivo segmental thickening, (2) cardiomyocyte elongation and dysfunction, and (3) increased focal protein phosphorylation on tyrosine residues. Interestingly, in a recent study using a swine model of tachycardia-induced heart failure, elongated myocytes with depressed contractile function were found to have alterations of cytoskeletal architecture,46 known to be regulated in various cell lines by the tyrosine kinase enzymatic system.30 47
By contrast, regions remote from the infarcted tissue show normal in vivo segmental thickening, elongated but normally contracting cells, and no signs of increased tyrosine kinase activity with respect to controls. Therefore, it is tempting to speculate that increased tyrosine kinase activity (or reduced tyrosine phosphatase activity) is associated with the combination of elongation and dysfunction rather than with myocyte elongation alone, perhaps reflecting a transition to myocyte failure in adjacent noninfarcted regions. Although from these data no causal relation between the two can be established, we believe that these observations raise additional interest in the study of this intracellular signaling pathway and the functional implications of different cellular hypertrophic responses.
The observed distribution of the tyrosine kinase substrates is optimal for a potential role in bidirectional communication between the intracellular and extracellular environments. Confocal microscopy demonstrated that the focal areas of phosphorylated tyrosine residues are contiguous with the inner aspect of the myocyte membrane, preferentially in correspondence with branching points and regions of cell-to-cell contact. The need for such an interfacing system has been postulated to explain the transduction of mechanical stretch signals through membrane receptors coupled to intracellular specific signaling pathways. The role of cellular mechanical transducers is thought to be played by membrane ion channels or receptors of the integrin family.48 Integrins, upon binding to specific extracellular matrix ligands, are able to activate the tyrosine kinase pathway, which, in turn, is thought to contribute to the hypertrophic response. In addition, intracellular calcium levels have been shown to facilitate the intracellular response to mechanical stretch, which ultimately leads to hypertrophy. Thus, potential mechanisms linking tyrosine kinase activation to myocyte hypertrophy and dysfunction exist, and their elucidation could provide further insight into our understanding of myocardial remodeling and failure.
The specific intracellular mechanisms responsible for reduced myocyte shortening in adjacent regions are not known. In the volume-overload model, both myocyte elongation and dysfunction are associated with a reduced relative volume of myofibrils and a depressed contractile response to increasing external Ca2+ concentrations.44 In addition, abnormalities accounting for mechanical dysfunction have been identified in cytosolic Ca2+ transients,49 myofilament composition,50 51 and contractile response to inotropic agents.52 Potential mechanisms of the myocyte dysfunction found in adjacent regions include an abnormal response to sympathetic stimulation52 or dysfunction in the process of excitation-contraction coupling,49 mediated by reduced Ca2+ availability or depressed myofilament sensitivity to intracellular Ca2+. Further investigation is needed to clarify the intracellular mechanisms responsible for myocyte failure during postinfarct remodeling.
Methodological Considerations
Left ventricular remodeling is a dynamic process in
which changes at the cellular level can be detected as early as a few
days after coronary occlusion in rats.43 Previous
experimental work showed that the rate of change depends on the animal
model but is generally maximal over the first weeks after infarction.
However, functional impairment and architectural changes progress
further over months.3 5 12 13 In this study, we chose to
assess ventricular remodeling at the single time point of 2
weeks after surgery; hence, comparisons with studies having different
time courses should consider time as a source of variability. Our
2-week time point was late enough for a reliable identification of the
scar tissue (complete infarct healing in the rat) and for the
development of significant ventricular remodeling,
documented by MRI as an increase in the left ventricular
endocardial area and endocardial area/myocardial wall area ratio.
The use of the MRI technique is relatively new in the rat model and required quite a high level of spatial and temporal resolution to characterize postinfarct left ventricular structure and function in such a small animal for a simultaneous assessment of global architecture and regional mechanical function in vivo. Nevertheless, the results of intraobserver and interobserver variability analyses were comparable to those calculated in previous studies using the same contouring and data analysis software on MRI images obtained from large-animal models and humans.
The assessment of isolated myocyte function was performed, for technical reasons, in conditions that diverge from physiological conditions in several aspects: temperature, [Ca2+] in the superfusate, and measurement of contractility in the unloaded state. Previous work demonstrated that myocyte populations with different contractility in the unattached, unloaded state show similar differences in contractility when the studies are performed in viscous media53 or after the cells are attached to a basement membrane substrate.44 In this regard, one could speculate that assessing cell contractility under conditions mimicking an external load might have evidenced differences even greater than those found. Also, for ethical reasons, the administration of sodium pentobarbital was routinely adopted at euthanasia: although a differential depressant effect of the anesthetic on the rat myocardium cannot be ruled out, there is no reason to believe that it would be the case.
We used monoclonal anti-phosphotyrosine antibodies and immunofluorescence to reveal focal areas of increased concentration of tyrosine kinase substrates in cardiomyocytes. The assessment of increased tyrosine kinase activity by other methods, although potentially more accurate and sensitive in ideal conditions, does not provide information on the intracellular localization of the source signal. Experimental factors inherent to this particular model, such as the small sample volumes and the inability to control for the contribution of nonmyocyte cells and damaged myocytes, would have impaired the reliability and sensitivity of bulk biochemical analytical techniques for the purposes of this study. As such, quantification of tyrosine-phosphorylated proteins on whole-cell lysates by SDS-PAGE and Western blot also proved to be unreliable for the presence of high background noise and wide animal-to-animal variations. Because immunocytochemistry combined with conventional fluorescence and confocal laser microscopy provided the capability to estimate the amount and the location of phosphotyrosine in the intact myocyte, we chose this approach to index tyrosine kinase activity.
Our study is in agreement with previous histomorphometric studies in infarcted rats that demonstrated greater myocyte elongation in the adjacent than in the remote regions. By contrast, we did not observe an increase in myocyte width as reported in previous experimental work on postinfarct remodeling.8 Although extensive remodeling was present in our model of large myocardial infarction as demonstrated in vivo by global left ventricular dilatation and increased volume-to-mass ratio, it is possible that greater myocardial damage would result in increased cell width secondary to higher intramural stresses early after infarction. Conversely, differences between our methods, assessing cell diameter in isolated cardiocytes, and those used in previous histological studies could have accounted for the different results. To examine this possibility, we performed measurements of cell diameter using the histological methods previously described.9 Both methods indicated no increase in cell width 2 weeks after coronary occlusion. The absence of increase in myocyte width is also in agreement with more recent studies54 showing that a significant increase of this parameter could be demonstrated only 4 weeks after myocardial infarction. Finally, one possible limitation of myocyte isolation techniques is that the cell populations may not be entirely representative of those originally present in the intact heart. In this regard, the agreement between the histological and isolated myocyte methods supports the reported results.
Myocyte Morphology and Ventricular
Remodeling
Our observations on global left ventricular function
after myocardial infarction are in good agreement with
echocardiographic longitudinal studies in the same
animal model of myocardial infarction that reported a reduction in
wall thickness of noninfarcted segments of the left ventricle relative
to chamber diameter.4 Ventricular remodeling
has recently been the object of numerous studies in an attempt to
identify the mechanisms responsible for the transition from
compensatory dilatation to ventricular dysfunction and
failure.1
We document an increase in myocyte length in cells from both the remote and adjacent regions secondary to the "in series" addition of new sarcomere units 2 weeks after myocardial infarction. This pattern of cellular response, characterized by myocyte slippage6 and elongation7 during postinfarct remodeling, has been attributed to the activation of Frank-Starling mechanisms to maintain cardiac output in the face of sudden myocardial loss.1 2 This entails increased levels of diastolic wall stress and mechanical stretch to accommodate progressively larger end-diastolic volumes. However, we also document a greater magnitude of myocyte elongation in cells isolated from adjacent regions, which is consistent with findings from previous studies that used different methods to assess cardiomyocyte length.8 9 Hemodynamically, this greater myocyte elongation found in the adjacent noninfarcted regions could be explained only by either greater mechanical stretch during diastole or greater stresses oriented along the long axis of myocytes in those regions during systole. In this regard, previous experiments in large-animal models of chronic postinfarct remodeling have demonstrated complex patterns of myocardial deformation during contraction in adjacent noninfarcted regions.55 These changes included a reduction in the magnitude of principal strains in those regions, with reorientation of myocardial deformation away from the radial direction.55 Alternatively, the exaggerated elongation of myocytes in adjacent regions could be secondary to humoral factors selectively active or potentiated in noninfarcted myocardium adjacent to the infarct border. Further studies are needed to elucidate the mechanisms involved in the regional heterogeneity of myocardial remodeling after infarction.
In conclusion, alterations intrinsic to the myocyte are associated with adjacent-region segmental dysfunction during postinfarct left ventricular remodeling. These alterations consist of greater cell elongation and depressed mechanical performance of myocytes isolated from adjacent noninfarcted regions compared with those isolated from regions located remote from the infarct border. In addition, myocytes from adjacent regions had an increased concentration of membrane-associated tyrosine phosphorylated proteins, suggesting an exaggerated activation of the tyrosine kinase pathway, known to be involved in both mechanical and humor-mediated cardiomyocyte hypertrophy. The greater myocyte elongation and dysfunction of adjacent noninfarcted regions may result from increased mechanical overload caused by the architectural alterations that characterize postinfarct left ventricular remodeling.
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Acknowledgments |
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This study was supported by Grant-in-Aid 92-10-26-01 from the National American Heart Association and a Grant-in-Aid from the Delaware Affiliate of the American Heart Association.
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Footnotes |
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1 References 3-93 4 5 6 7 8 9 , 1212 , 41-4341 42 43 , 4949 , 5151 , 5252 , 5454 .
Received May 8, 1995; revision received October 30, 1995; accepted November 1, 1995.
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