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© 1998 American Heart Association, Inc.
Original Contributions |
Angiotensin II Stimulation In Vitro Induces Hypertrophy of Normal and Postinfarcted Ventricular Myocytes
From the Department of Medicine, New York Medical College, Valhalla.
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Abstract |
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Abstract—To determine whether angiotensin II (Ang II) stimulation of adult ventricular myocytes in vitro results in cellular hypertrophy, the changes in myocyte volume and protein content per cell were examined by confocal microscopy. Moreover, the possibility was considered that the upregulation of Ang II receptors on myocytes after infarction may potentiate and/or accelerate Ang II–mediated myocyte growth. Left ventricular myocytes isolated from control and failing hearts 3 days after infarction were cultured for 3 and 7 days in the presence of Ang II. Normal myocytes did not show an increase in volume and protein content at 3 days, but a 16% and 20% increase in these respective parameters was found at 7 days. Cell growth was faster and greater in myocytes from postinfarcted hearts. In these cells, myocyte volume increased 23% and protein content increased 28% at 3 days after Ang II administration. The higher hypertrophic reaction of myocytes from infarcted hearts occurred in spite of a 19% larger volume at isolation. In both groups of myocytes, the AT1 receptor blocker losartan completely inhibited the consequences of Ang II. Conversely, the AT2 receptor antagonist PD123319 had no effect on Ang II–induced hypertrophy. In conclusion, Ang II promotes myocyte growth through the activation of AT1 receptors, which modulate the time and magnitude of this cellular response.
Key Words: angiotensin II • myocardial infarction • confocal microscopy • myocyte growth • protein content
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several in vitro studies have documented the fact that neonatal cardiac myocytes possess the various components of the renin-angiotensin system.1–3 Sarcomere stretching, applied in an attempt to mimic the in vivo condition of diastolic overloading, is coupled with the cellular release of angiotensin II (Ang II) and the activation of myocyte cellular hypertrophy.4 However, whether Ang II can promote hypertrophy of adult ventricular myocytes is still controversial.5,6 Angiotensin-converting enzyme (ACE) inhibitors prevent myocardial hypertrophy in the overloaded heart,7 but it is unclear whether this negative trophic effect is due to reduction in ventricular loading, attenuation in the generation of Ang II systemically and locally, or a combination of these mechanisms. The possibility has also been advanced that ACE inhibitors may exert their influence on myocyte growth indirectly through the preservation of bradykinin.8 Indices of a modest anabolic response of adult feline myocytes to Ang II have been claimed in in vitro preparations.5 However, changes in myocyte cell volume were not measured, raising the question of the actual impact of Ang II on cell growth.
Adult rat ventricular myocytes express Ang II AT1 receptors,9 and radioligand binding assays have identified surface AT1 receptors on these cells.10 Conversely, the functional significance of Ang II binding sites on mature myocytes has been questioned, challenging the contention that Ang II may be capable of stimulating growth and mechanical responses.5,6,11 Similar reservations have been made for neonatal myocytes.12 However, this view is not consistent with in vivo studies demonstrating that Ang II receptors on myocytes increase shortly after coronary artery occlusion, possibly modulating the reactive growth adaptation of the remaining viable cells in the infarcted heart.10 In this regard, AT1 receptor antagonists attenuate cardiac hypertrophy in this model.13 AT2 receptors may also be expressed in the overloaded myocardium,14,15 and this may interfere with the growth-promoting action of AT1 receptors.16 The present study sought to determine whether Ang II stimulation of normal adult ventricular myocytes in vitro would result in cellular hypertrophy characterized by changes in myocyte volume and protein content per cell. Moreover, the hypothesis was advanced that the upregulation of Ang II receptors on myocytes after infarction10,17 might be coupled with a potentiation and/or acceleration of Ang II–mediated cellular hypertrophy.
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Materials and Methods |
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Coronary Artery Occlusion
Twenty-four male Sprague-Dawley rats (Charles River Breeding Laboratories, North Wilmington, Mass), weighing
250 g, were used for
the surgical induction of myocardial infarction. With the animals under
ether anesthesia, thoracotomy via the third left
intercostal space was performed, the heart was exteriorized, and the
left coronary artery was ligated 1 to 2 mm from its
origin. The chest was closed, pneumothorax was reduced by negative
pressure, and the animals were allowed to recover. Nine infarcted rats
died shortly after the operation. Fifteen sham-operated rats were
treated similarly, except that the ligature around the coronary
artery was not tied.10,18 The sham operation
resulted in no mortality. Animals were killed 3 days after
coronary artery occlusion or sham operation.
Global Cardiac Performance
Animals were anesthetized with chloral hydrate (300
mg/kg body wt IP), and the external right carotid artery was exposed
and cannulated with a microtipped pressure transducer catheter (Millar
SPR-249) connected to an electrostatic chart recorder (Gould ES
2000). After arterial blood pressure was monitored, the
catheter was advanced into the left ventricle for the evaluation of
left ventricular pressures, +dP/dt, and -dP/dt.
Subsequently, a second catheter (Millar SPR-595), with a 120° curved
tip, was inserted in the right jugular vein and advanced through the
superior vena cava and the right atrium into the right
ventricular chamber for the measurements of central venous
pressure, right ventricular pressures, and dP/dt. Thus,
measurements were made of systemic arterial and venous
blood pressures, ventricular pressures, and dP/dt in the
closed-chest preparation.9,10,18
Myocyte Isolation
At the end of the hemodynamic determinations,
hearts were rapidly excised, and myocytes from the left ventricle were
enzymatically dissociated.9,10,18 Hearts were
placed on a stainless-steel cannula for retrograde perfusion through
the aorta. The solutions were supplements of modified commercial
Joklik's MEM (JRH Biosciences). HEPES-MEM contained (mmol/L) NaCl 117,
KCl 5.7, NaHCO3 4.4,
KH2PO4 1.5,
MgCl2 17, HEPES 21.1, glucose 11.7,
L-glutamine 2, and taurine 10 in addition to amino acids,
vitamins, and 21 mU/mL insulin and was adjusted to pH 7.2 with NaOH.
This solution was 292 mOsm, isosmolar with rat serum. Resuspension
medium was HEPES-MEM supplemented with 0.5% BSA and 0.3 mmol/L
calcium chloride, adjusted to 292 mOsm. The cell isolation procedure
consisted of 3 main steps: (1) Calcium-free perfusion: Blood washout
and collagenase (selected type II, Worthington Biochemical)
perfusion of the heart was carried out at 34°C with HEPES-MEM gassed
with 85% O2/15% N2. (2)
Mechanical tissue dissociation: After removal of the heart from the
cannula, the left ventricle inclusive of the septum was separated and
minced. In infarcted hearts, only the spared portion of the ventricle
was used. Collagenase-perfused tissue was subsequently
shaken in resuspension medium containing collagenase and
0.3 mmol/L calcium chloride. Supernatant cell suspensions were
washed and resuspended in resuspension medium. (3) Separation of intact
cells: Intact cells were enriched by centrifugation and
by discarding the supernatant. This procedure was repeated 4 or 5 times
in each preparation to remove nonmyocyte cells, cell debris,
and the residual collagenase. Each
centrifugation was performed at 30g for 3
minutes. Rectangular, trypan blue–excluding cells constituted nearly
80% of all myocytes. The average number of myocytes obtained from the
left ventricle of sham-operated control rats and infarcted rats was
6x106 and 3.5x106,
respectively. The contribution of interstitial cells was
assessed by counting 1000 cells in each left ventricle and then
computing from these counts the respective fractions of myocytes and
nonmyocytes encountered. Consistent with previous
results,9,10 nonmyocytes accounted for
<1% of the cell population.
Cell Culture
Myocytes were plated in Petri dishes (Corning), coated with 0.5
µg/cm2 of laminin (Sigma Chemical Co), at a
density of 2x104 cells/cm2.
Cells were incubated in serum-free medium (SFM), consisting of Eagle's
MEM with nonessential amino acids (Sigma), supplemented with penicillin
(100 U/mL), streptomycin (50 µg/mL), transferrin (10 µg/mL), and
BSA (0.1%). Cultures were incubated at 37°C in an atmosphere
containing 5% CO2. SFM was changed 30 minutes
after plating to remove myocytes that did not attach to the dish.
Concurrently, Ang II at 10-9
mol/L19,20 was added in HBSS. An equal volume of
HBSS was added to control cultures. Myocytes were exposed to Ang II for
3 and 7 days. Pretreatment with losartan (Merck) at
10-7 mol/L and PD123319 (Parke-Davis
Pharmaceutical Co) at 10-7 mol/L was
accomplished by adding these drugs 30 minutes before exposure of cells
to Ang II. Specifically, PD123319 at a concentration of
10-3 mol/L was dissolved in 100 mmol/L
Tris-HCl. The medium, Ang II, losartan, and PD123319 were
changed daily. At completion, cells were washed with cold HBSS and were
fixed in 10% phosphate-buffered formalin for 15 minutes. Phenylalanine
incorporation (10 µCi/mL) was measured in myocytes isolated 3 days
after infarction. Cells were cultured in SFM for 24 hours;
subsequently, Ang II and phenylalanine were added. Three concentrations
of Ang II were used: 10-11, 10-9, and
10-7 mol/L. After 24 hours, samples were washed 3 times
with HBSS supplemented with 10 mmol/L cold phenylalanine and were
fixed with 10% trichloroacetic acid for 1 hour at 4°C. Cells were
again washed 3 times with 95% ethanol and redissolved with 1 mL of
0.1N NaOH. These aliquots were used for scintillation counting. Eight
separate determinations were performed using 8 distinct cell
isolations.
Western Blot
For immunoblot assay of AT1
receptors, myocytes were lysed with 150 to 200 µL of lysis buffer
containing the protease inhibitors phenylmethylsulfonyl
fluoride (2 mmol/L), aprotinin (1 µg/mL), dithiothreitol
(5 mmol/L), and Na3VO4
(1 mmol/L). Equivalents of 100 to 125 µg of protein were
separated by 10% SDS-PAGE. Proteins were transferred on nitrocellulose
filters and exposed to rabbit polyclonal anti-human
AT1 receptor antibody (No. 306, 5 µg/mL, Santa
Cruz). Bound antibodies were detected by peroxidase-conjugated
anti-rabbit IgG and ECL reagents (Amersham). AT1
receptor was detected as a 41-kDa band. This analysis included
left ventricular myocytes isolated from 5 sham-operated and
5 infarcted rats killed 3 days after surgery. Western blotting was
performed at 6 hours and 3 days after plating.
Structural Properties of Myocytes
Myocyte dimensions were measured with a computerized image
analysis system (Jandel Scientific): 100 to 200 binucleated
myocytes from each preparation were examined to collect length, width,
and area. Moreover, cell volume was derived from these geometric
parameters. Cells in cultures assume a cross-sectional
area, which resembles a flattened ellipse. The ratio of the minor axis
(b) to the major axis (a) of the ellipse was obtained by measuring
these parameters in 20 myocytes in each preparation by
confocal microscopy (Bio-Rad MRC-1000). Cell volume
(VC) was calculated assuming an elliptical cross
section with a major axis that was equivalent to cell width and a minor
axis that was computed from the measured ratios. Cell length (L) was
measured directly21–23:
VC=[
· (a/2) · (b/2)]L.
The quantitative analysis described above was complemented in some experiments with a direct evaluation of the volume of binucleated myocytes by confocal microscopy. For this purpose, myocytes were stained with FITC (1 µg/mL) for 30 minutes at room temperature to visualize the cell cytoplasm and with propidium iodide to label the nuclei. By optical section reconstruction of the entire cell in the z or y plane, the volume of sections, 1 µm apart, was collected, and their sum was calculated to yield the total cell volume. Twenty myocytes in each preparation were analyzed in this manner. These cells were randomly sampled by including only binucleated myocytes vertically oriented in the microscopic field.21–23
Cell Distribution
The distribution of myocytes in each experimental condition was
divided according to number of cells in an established range of
lengths, cross-sectional areas, and volumes. The range of length used
varied from 30 to 150 µm. Histogram buckets were established
with a size of 3 µm, and frequency distribution histograms were
constructed by plotting the number of cells on the ordinate and the
cell length on the abscissa. A similar approach was followed for the
analysis of the distribution of cell cross sections and
volumes. The range of cross-sectional areas used varied from 100 to
1500 µm2, and the histogram bucket was
40 µm2. Finally, the range of cell volume
used varied from 8000 to 100 000 µm3, and
the histogram bucket was 2000 µm3.
Measurement of Protein Content per Cell
Formalin-fixed myocytes were incubated in PBS containing FITC
(0.1 µg/mL), propidium iodide (10 µg/mL), and RNase A (1 mg/mL) for
30 minutes at room temperature. After staining, cultures were washed in
PBS and embedded in Vectashield (Vector Laboratories) to prevent
photobleaching during the measurements. Total fluorescence of
individual myocytes, which corresponded to the protein content per
cell,24,25 was determined by confocal microscopy.
The intensity of FITC fluorescence was measured by optically
sectioning the entire thickness of each myocyte and recording
the intensity of fluorescence in each of these sections. These
intensities were added to yield the total fluorescence in each
myocyte. Staining with propidium iodide was used to identify
binucleated myocytes. Fifty myocytes in each culture were measured in
this manner.
Data Analysis
All measurements are presented as mean±SD computed from
the average results obtained from each culture; n values for each
determination, which correspond to the number of independent cultures,
are listed in the text or in the legend to each figure. Comparisons
between 2 values were performed by the unpaired Student t
test. Statistical significance in multiple comparisons among
independent groups of data, in which ANOVA and the F test indicated the
presence of significant differences, was determined by the Bonferroni
method.26 Values of P<0.05 were
considered to be significant.
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Results |
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Myocardial Infarction and Cardiac Function
Myocardial infarction at 3 days did not alter body weight (sham-operated rats, 285±30 g, n=15; infarcted rats, 264±35 g, n=15) and heart weight (sham-operated rats, 923±99 mg; infarcted rats, 939±120 mg). However, infarction was characterized by a 3.0-fold (P<0.001) increase in left ventricular end-diastolic pressure and a 30% (P<0.00l) decrease in left ventricular systolic pressure (Figure 1
). Similarly, left
ventricular developed pressure was decreased 45%
(P<0.001), from 106±6 to 58±13 mm Hg. Moreover,
left ventricular +dP/dt was reduced 41%
(P<0.00l) and -dP/dt was reduced 40%
(P<0.001) in infarcted hearts. Measurements of right
ventricular hemodynamics (Figure 1) showed
that right ventricular end-diastolic pressure
was elevated 4.5-fold (P<0.001), whereas right
ventricular systolic pressure and
diastolic pressure did not change significantly after
infarction. In contrast, +dP/dt was reduced 18% (P<0.005),
and central venous pressure was increased 5.7-fold
(P<0.001). In summary, myocardial infarction resulted in
left ventricular failure and right ventricular
dysfunction.
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Ang II and Adult Ventricular Myocytes in
Culture
A dose of 10-9 mol/L Ang II was
selected. This concentration was established by performing preliminary
studies in postinfarcted myocytes at 24 hours in culture in SFM. These
cells were exposed to 3 different doses of Ang II (10-7,
10-9, and 10-11
mol/L) in the presence of
[3H]phenylalanine (10 µCi/mL). Radioactivity
measurements were made 24 hours later. Phenylalanine incorporation per
1000 myocytes was 1882±198 dpm (n=8), 1828±163 dpm (n=8), 1694±152
dpm (n=8), and 1522±178 dpm (n=8) at 10-7,
10-9, and 10-11 mol/L and
in the absence of Ang II, respectively. The radioactivity values
obtained with 10-7 and
10-9 mol/L Ang II were not statistically
different. In comparison with baseline, 10-9
mol/L Ang II produced a 20% (P<0.01) increase in protein
synthesis. On this basis, a 10-9 mol/L Ang II
dose was used in all experiments.
The effects of Ang II on cellular hypertrophy were
evaluated in binucleated myocytes because they represent 90%
to 95% of muscle cells of the left ventricle.27
A relevant problem in the analysis of the impact of Ang II on
myocytes in vitro concerned the changes in the morphological
characteristics of the cells. Myocytes were cylindrical in shape at the
time of isolation and plating, but this property was not maintained by
the majority of cells in culture. The cell population consisted of a
mixture of rod-shaped and rounded myocytes (Figure 2A and 2B). Moreover, the proportion of
rectangular and rounded cells changed from 2 hours after plating to 3
and 7 days in culture. These values in control myocytes were as
follows: plating=91.2±2.9% (n=4), 3 days=58.5±6.1% (n=5), and 7
days=49.3±14.4% (n=6). Corresponding values in myocytes after
infarction were as follows: plating=89.7±4.6% (n=4), 3 days=55.8±8%
(n=5), and 7 days=52.0±8.5% (n=6). At 3 and 7 days, most rectangular
cells possessed long thin projections that altered the normal
appearance of these myocytes (Figure 2C and 2D), complicating even
further the morphometric assessment of cell volume by standard
techniques. Since loss of cylindrical configuration was a phenomenon
that affected a large fraction of myocytes, cell size was measured
separately in rod-shaped and rounded myocytes (see below). This was
done to establish whether modifications in cell shape influenced the
growth response of myocytes to Ang II stimulation in vitro. Cells
maintained in SFM showed changes in the cytoplasmic composition with
reduction in the myofibrillar compartment. Although lesser in
magnitude, similar aspects have also been observed in the presence of
Ang II.
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An additional factor in these in vitro experiments involved the loss of contraction of myocytes. Shortly after plating, 80±6% (n=4) of the normal cells in SFM were contracting, but this activity was restricted to 1.3±0.2% (n=4) and 0.4±0.2% (n=4) of the myocytes at 3 and 7 days, respectively. A single daily administration of Ang II at 10-9 mol/L did not increase the fraction of contracting myocytes: 3 days=1.1±0.2% (n=4) and 7 days=0.5±0.3% (n=4). Ventricular myocytes isolated from infarcted hearts behaved in a similar manner. Under SFM conditions, 75±9% (n=4) of the cells were contracting at plating, with 1.3±0.4% (n=4) contracting at 3 days and 0.4±0.1% (n=4) contracting at 7 days. After Ang II administration, values at 3 and 7 days were 1.2±0.2% (n=4) and 0.3±0.3% (n=4), respectively. In summary, adult ventricular myocytes changed their configuration and lost their ability to contract in culture, and Ang II did not modify these phenomena.
Myocyte Cell Volume
The alterations of myocyte shape with time in culture required a
complex approach for the evaluation of myocyte volume. This
parameter was obtained by the product of myocyte cell
area (measured by an image analysis system) and cell thickness
(obtained by confocal microscopy). Optical sections of myocytes on the
z plane by confocal microscopy were used to determine the magnitude of
cell flattening (Figure 3). The ratios of
cell thickness to cell width are listed in Table 1; greater degrees of
flattening were associated with lower ratios. These values were
obtained by measuring 20 myocytes in each preparation. In each culture
dish, myocyte cell area and width were measured randomly in a minimum
of 100 cells to a maximum of 200 cells with an image analyzer.
These data, in combination with the cell thickness–to–cell width
ratio, were used to compute the volume, cross-sectional area, and
length of each cell in this larger myocyte sampling (Figure 4).
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A second methodology was used to evaluate myocyte cell volume in these
experiments. Figure 5A illustrates 12
optical sections, 1 µm apart, of a control myocyte kept in SFM
for 7 days. By this 3-dimensional section reconstruction in the y
plane, myocyte volume was assessed and, in this specific case, was
found to be 18 000 µm3. Myocyte
cross-sectional area and length were 182
µm2 and 99 µm, respectively. An
identical determination in a myocyte isolated from an infarcted
ventricle and stimulated in vitro by Ang II for 3 days is depicted in
Figure 5B. In this cell, optical sectioning included 16 images, 1
µm apart, and myocyte volume, cross-sectional area, and length were
37 400 µm3, 367
µm2, and 102 µm, respectively.
Additionally, absolute values of myocyte characteristics were obtained
and compared by evaluating the same cell in the y and z planes. For
example, the control myocyte shown in Figure 5C and 5D, which was
cultured in SFM for 3 days, had the following values: in the y
orientation, cell length, cross-sectional area, and volume were 68
µm, 244 µm2, and 16 575
µm3, respectively. Corresponding values in the
z plane were 66 µm, 247 µm2, and
16 320 µm3. The similarity in these
parameters suggested that one evaluation on the y or z
plane was sufficiently accurate for the assessment of myocyte cell
volume. This approach, however, is very time consuming and was
restricted to 20 myocytes in each preparation. In summary, a
combination of light and confocal microscopy was used for the
morphometric analysis of the size and shape of
ventricular myocytes in culture.
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Ang II and Myocyte Volume in Control Cells
Figure 6A illustrates the
average volume of control myocytes at plating and after exposure to SFM
or Ang II for 3 and 7 days. Cells kept in SFM showed no change in
volume at 3 days and an 11% (P<0.05) decrease at 7 days.
In comparison with myocytes maintained in SFM, Ang II administration
resulted in a 16% (P<0.001) increase in mean cell volume
at 7 days. The 3% increase at 3 days did not reach statistical
significance. Ang II was capable of preventing the reduction in myocyte
size that occurred in SFM with time. As also shown in Figure 6A, the
AT1 receptor blocker, losartan, inhibited
Ang II–mediated myocyte growth, whereas the AT2
receptor antagonist PD123319 had no influence on myocyte
volume.
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P<0.05 vs
value at plating.
The results in Figure 6A
were obtained by combining measurements in
rod-shaped and rounded myocytes. This was done because comparable
values were found in these 2 cell groups in SFM (3 days:
rod-shaped=24 530±1299 µm3, n=5;
rounded=23 488±1869 µm3, n=5
[P=0.34]; 7 days: rod-shaped=20 697±1791
µm3, n=6; rounded=21 391±1972
µm3, n=6 [P=0.58]) and after Ang
II stimulation (3 days: rod-shaped=24 162±1647
µm3, n=5; rounded=24 803±1939
µm3, n=5 [P=0.59]; 7 days:
rod-shaped=24 206±1424 µm3, n=6;
rounded=24 034±1484 µm3, n=6
[P=0.85]). Moreover, similar values in rod-shaped and
rounded cells were observed in the presence of losartan and
PD123319 (data not shown). The direct evaluation of myocyte volume of
rod-shaped cells by confocal microscopy is listed in Table 2. Ang II–treated myocytes at 7 days were 18% larger than
the corresponding cells kept in SFM, and this difference was
significant (P<0.005). In summary, Ang II promoted myocyte
hypertrophy through the activation of the
AT1 receptor subtype.
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Ang II and Cell Volume of Postinfarcted Myocytes
Figure 6B illustrates that myocytes from infarcted
ventricles underwent some atrophy when placed in SFM. Myocyte volume
was reduced by 3% (P=NS) at 3 days and 13%
(P<0.01) at 7 days. In comparison
with control cells maintained in SFM, the administration of Ang II
resulted in a 23% (P<0.005) increase in myocyte volume at
3 days. At this interval, Ang II–treated myocytes were 18%
(P<0.05) larger than freshly isolated cells at plating.
Seven days of Ang II stimulation resulted in a 15%
(P<0.05) increase in myocyte volume, although this
parameter was essentially identical to that of cells at
plating. Losartan blocked Ang II–induced myocyte
hypertrophy at 3 and 7 days, whereas PD123319 did not
interfere with this response (Figure 6B). Measurements of cell size by
confocal microscopy in subsets of myocytes confirmed these results
(Table 3).
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When the data in Figure 6 were compared, it was apparent that myocytes
from infarcted hearts were 19% (P<0.05), 13%
(P<0.05), and 16% (P<0.001) larger in volume
than cells from sham-operated animals at plating and at 3 and 7 days in
SFM, respectively. The addition of Ang II increased this difference at
the earlier interval in culture, since cell volume was 35% greater in
postinfarcted myocytes than in normal cells at 3 days
(P<0.001). However, at 7 days after Ang II administration,
the variation in cell volume between these two groups of myocytes was
15% (P<0.05).
To determine whether the effects of Ang II on normal and postinfarcted myocytes involved the entire cell population or a fraction of myocytes, the changes in the distribution of length, cross-sectional area, and volume of these cells were measured. This analysis included 1540 control cells maintained in SFM for 7 days and 1540 control myocytes exposed to Ang II for the same period. The 7-day interval was selected because no myocyte hypertrophy was found with Ang II at 3 days. Conversely, myocytes from infarcted ventricles were examined at 3 days, since hypertrophy was essentially completed at this time and decreased from 3 to 7 days; 1680 myocytes kept in SFM and 1680 cells treated with Ang II were evaluated.
Figure 7 illustrates the ranges of muscle
cell lengths, cross-sectional areas, and volumes observed in control
myocytes kept in SFM and after Ang II stimulation. In SFM, the
distributions of cell cross section and volume were evenly balanced
around the mean values, with the majority of cells located close to the
means. Ang II treatment modestly shifted to the right the distribution
of myocyte cross-sectional areas and volumes. The values of myocyte
length were more variable in both groups of cells, and Ang II
produced a small change in this parameter. The 1540
myocytes kept in SFM and 1540 cells exposed to Ang II were subsequently
used to compute the average values of length, cross-sectional area,
transverse diameter, and volume. A 16% increase in myocyte volume,
from 20 942±7718 to 24 391±9189 µm3,
was found, and this was the result of a 13% (P<0.001)
increase in cross-sectional area, from 409±147 to 463±175
µm2, and a 2.4% (P<0.05)
lengthening of the cells, from 53.2±16.3 to 54.5±15 µm. Ang II
produced a 6.2% (P<0.001) expansion in transverse
diameter, from 22.43±4.24 to 23.83±4.59 µm. This implies that
the lateral dimension of myocytes increased 2.6-fold more than the
longitudinal axis of the cells.
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An identical analysis was performed with myocytes from
infarcted hearts. After Ang II administration, there was a change in
the cell length distribution, and a comparable phenomenon was noted
with respect to myocyte cross-sectional area and volume (Figure 8). Growth promoted by Ang II resulted in
a broader lowered peak and extended to the right curve, which was more
apparent than in control myocytes. Average myocyte volume increased
20% (P<0.001), from 27 274±11 173 to
32 682±17 172 µm3. Changes in myocyte
length, 8.4% (P<0.00l), from 59.98±21.62 to
65.03±25.01 µm, and cross-sectional area, 9.7%
(P<0.001), from 479±192 to 526±244
µm2, were comparable in Ang II–treated cells.
Transverse diameter increased 4.3% (P<0.001), from
24.23±4.84 to 25.27±5.69 µm, which was nearly 50% smaller
than the expansion in length of myocytes. A relevant aspect illustrated
in Figures 7 and 8 is that the changes in cell volume in each cell
group involved only a fraction of the population. The peak of the
histogram was lower than in the corresponding SFM control, and the
cells were shifted to the right toward higher values. In contrast, the
left side of the histogram and the position of its peak remained
essentially unchanged. This may imply a partial nonuniform adaptation
of myocytes to Ang II. In summary, Ang II resulted in cellular
hypertrophy of postinfarcted myocytes, and this response
occurred earlier and was of greater magnitude than in control
cells.
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Expression of AT1 Receptor in Myocytes
This analysis was performed to establish whether the
difference in Ang II receptor density previously shown between control
and postinfarcted myocytes in vivo10,17 persisted
in culture under SFM conditions. The in vitro maintenance of a
higher level of AT1 receptor protein in myocytes
surviving an acute myocardial infarction should have influenced the
effects of Ang II stimulation on cellular growth. Figure 9 illustrates the changes in
AT1 receptor protein in myocytes obtained from
sham-operated and infarcted rats after 6 hours and 3 days in culture in
SFM. Postinfarcted myocytes had higher levels of expression of
AT1 receptors than did control cells at both time
intervals examined. Densitometrically, there was a 1.8-fold increase at
6 hours (OD: control cells=29±5, n=5; postinfarcted cells=52±12, n=5;
P<0.005) and a 1.7-fold increase at 3 days (OD: control
cells=34±7, n=5; postinfarcted cells=58±14, n=5; P<0.01).
However, the small differences in each cell group between 6 hours and 3
days in culture were not statistically significant. In summary,
AT1 receptor protein was higher in postinfarcted
myocytes than in control cells in culture.
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Ang II and Protein Content per Cell
To confirm the ability of Ang II to induce myocyte
hypertrophy, the amount of protein per cell was determined
by confocal microscopy after staining with FITC. This technique results
in the labeling of proteins, and small differences in
fluorescence intensity can be detected
accurately.24 Ang II increased this
parameter 1% (P=NS) and 20%
(P<0.00l) in control myocytes at 3 and 7 days, respectively
(Figure 10). In postinfarcted myocytes,
a 28% increase (P<0.00l) at 3 days and a 20% increase
(P<0.05) at 7 days in protein content per cell was noted
after Ang II administration. Losartan inhibited the effects of
Ang II on both myocyte populations. In contrast, the
AT2 receptor blocker PD123319 did not influence
the changes produced by Ang II (Figure 10). In summary, Ang II
increased the protein level of myocytes, but this phenomenon took place
earlier and was enhanced in postinfarcted cells.
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of the present study indicate that Ang II in the nanomolar range was capable of inducing hypertrophy of adult ventricular myocytes in culture in the absence of cellular loading and contractile activity. This response was demonstrated quantitatively by measurements of myocyte volume and protein content per cell. Changes in myocyte size and protein levels were documented by confocal microscopy. Left ventricular myocytes from infarcted hearts showed earlier increases in cell volume and protein content per cell than did control myocytes. Moreover, enlarged postinfarcted myocytes achieved a greater additional magnitude of hypertrophy, suggesting that the growth-promoting effects of Ang II were enhanced in cells surviving an acute myocardial infarct. These adaptations were coupled with activation of surface AT1 receptors only. AT2 receptors appeared not to be involved in Ang II–mediated myocyte growth.
Ang II and Myocyte Growth
In the last several years, numerous in
vivo7,9,28–30 and in
vitro1–6 studies have characterized the
influence of Ang II on ventricular myocytes. Ang II may
induce 3 different responses: stimulation of myocyte
hypertrophy,2,5,16 improvement in the
mechanical behavior and Ca2+ transients of the
cells,31–33 and activation of myocyte
apoptosis.19,20,34 However, the ability
of Ang II to affect myocyte
contractility11 and cell
growth5,6,12 has been repeatedly challenged in
neonatal and adult myocytes. Since the impact of Ang II on myocardial
function has been documented in vivo in several model
systems35 and in isolated papillary
muscles,33 the negative observations at the
cellular level raise questions about the validity of the preparations.
Similarly, the anabolic consequences of Ang II on myocytes have been
criticized on the basis of semiquantitative methods that provide no
information about the actual changes in myocyte
volume.12 Additionally, alterations in protein
content per cell were not measured, and conclusions were drawn on
degrees of amino acid incorporation, neglecting the role of Ang II on
the catabolic and anabolic states of myocytes over time. Conversely,
moderate increases in myocyte protein have been shown in adult
quiescent myocytes from normal cats 7 days after the addition of Ang
II,5 although myocyte cell volume was not
obtained.
Observations in the present study are consistent with a growth-stimulating capacity of Ang II on ventricular myocytes. Cellular hypertrophy was measured by confocal microscopy, which allowed comparisons between changes in size and protein content in the same cells. Although only binucleated myocytes were analyzed in the present study, this approach permits the evaluation of the volume of mononucleated and multinucleated cells as well.21–23 At present, this information can be obtained only by this technique. In the present experiments, cell growth was faster and greater in myocytes from failing infarcted hearts. In spite of a 19% larger volume of freshly isolated myocytes from infarcted hearts, Ang II produced a 40% to 45% higher hypertrophic response in these cells than in control myocytes. The concomitant increase in cytoplasmic protein and myocyte dimension unequivocally indicated cell growth and not dedifferentiation. This latter phenomenon is characterized by a reduction in protein concentration per cell.35,36 Importantly, measurements of cell volume by confocal microscopy are not influenced by changes in cell shape.37 This methodology permits a direct evaluation of cell size.
Some additional comments concerning the interpretation of these findings are in order. The discussion above is based on the changes in volume of myocytes maintained in SFM in the absence and presence of Ang II. However, the lack of Ang II was characterized by a reduction in size of cells from control and postinfarcted hearts, indicating that adult quiescent myocytes in culture are subject to cellular atrophy. From plating to 7 days, the degree of this phenomenon averaged 12% in both groups of myocytes. Seven days of Ang II essentially maintained the original volume of myocytes isolated from sham-operated rats, questioning whether cellular hypertrophy actually occurred. Conversely, postinfarcted myocytes, stimulated for 3 days with Ang II, reached a volume that was not only 23% greater than that for the cells in SFM but also 18% larger than that for myocytes at plating, demonstrating an absolute increase in cell size.
These results were surprising and difficult to interpret. However, quiescent myocytes in vitro may be expected to undergo atrophy, mimicking a condition repeatedly shown in vivo in the presence of attenuated39 or abolished myocardial loading.40,41 Myocytes in SFM are unloaded and do not exhibit contractile activity; these characteristics have been shown to influence the structural organization and relative amount of the myofibrillar compartment of the cytoplasm.42 Undifferentiated areas of myocyte cytoplasm, characterized by limited myofibrillar structures, have been found in cells maintained in SFM, resembling the morphological aspects of ongoing myocyte atrophy.40 Although in a more restricted manner, similar aspects have been detected in myocytes exposed to Ang II, confirming that cellular atrophy occurs in vitro. Thus, Ang II may be capable of producing a significant growth response in myocytes, which may be obscured by cellular atrophy under culture conditions.
Why Ang II not only induces cellular hypertrophy but also stimulates myocyte apoptosis is complex.19,20 Cell death is restricted to a small percentage of the population,20 whereas cell growth involves a larger fraction of myocytes. This differential response of adult rat ventricular myocytes to Ang II may reflect heterogeneity in the expression of genes that protect cells from apoptosis or facilitate this process.43 For example, the low level of apoptosis detected in the failing canine heart is associated with an increase in the number of cells labeled by p53.44 This transcription factor is activated by Ang II,45 and this may lead to a reduction in the Bcl-2/Bax protein ratio in the cytoplasm, enhancing the susceptibility of cells to trigger their suicide program.43
AT1 and AT2 Receptor Subtypes and
Myocyte Hypertrophy
Ventricular myocytes possess 2 classes of
pharmacologically distinct and functionally active surface Ang II
receptors. Neonatal cardiac myocytes exhibit both
AT1 and AT2 receptor
subtypes,46,47 and each subtype accounts for 50%
of the specific binding.46 Additionally,
mechanical stretch in vitro increases the expression of
AT1 and AT2 receptors by
3-fold.48 Similarly, pathological states of
the adult heart characterized by sudden increases in
diastolic wall stress and myocyte stretching result in
upregulation of Ang II receptors. Such a response occurs acutely after
infarction, in which the density of Ang II receptors on the spared
myocytes increases by nearly 50% and 100% at 3
days17 and 7 days,10
respectively. Ventricular dysfunction induced by
nonocclusive coronary artery constriction typically shows a 3-
to 4-fold augmentation in Ang II receptors on
myocytes.9 However, control myocytes exhibit
AT1 receptors only,10 and
the existence and relative contribution of AT1
and AT2 receptor subtypes were not examined under
these experimental conditions. The possibility of an upregulation of
AT2 receptors on myocytes has been raised in
pressure-overload hypertrophy14 and
in the postinfarcted heart.49 Unfortunately, the
use of myocardial tissue for the preparation of cellular membranes
failed to provide unequivocal evidence of surface
AT2 receptor on stressed myocytes.
Results in the present study demonstrate that Ang II–induced myocyte hypertrophy was mediated by the activation of AT1 receptors, since the AT1 antagonist losartan completely inhibited cellular growth and the increase in protein content per cell. Moreover, AT1 receptor protein was increased in postinfarcted myocytes, which exhibited a greater Ang II–mediated growth response. Conversely, the AT2 receptor blocker PD123319 failed to modify the effects of Ang II on myocyte hypertrophy. These phenomena were apparent in both control myocytes and myocytes isolated from infarcted hearts. However, the increase in Ang II receptor protein on the viable cells after infarction was associated with an earlier and greater reactive response, pointing to the critical role of the number of AT1 binding sites in the modulation of cellular growth. The prevailing increase in myocyte length induced by Ang II in postinfarcted myocytes raises the possibility that the growth adaptation obtained in this cell population in vitro maintains the characteristics detected in vivo. In this regard, the elevation in diastolic load on the surviving myocardium after infarction in rats and humans is associated with a predominant lengthening of myocytes, which contributes to ventricular dilation.30 ACE inhibitors reduce myocyte hypertrophy and cavitary volume after infarction.30 The nonuniform shift to higher values in the distribution of cell volumes in Ang II–stimulated cultures in the present study strongly suggests that the myocyte population consisted of Ang II–responding and nonresponding cells. The enzymatic dissociation of myocytes with collagenase may alter receptor sites, affecting the capability of cells to perceive the effects of Ang II. This potential artifact may be responsible for the contrasting findings concerning the influence of Ang II on the growth of neonatal and adult ventricular myocytes. Importantly, this technical defect may result in an underestimation of the actual magnitude of myocyte hypertrophy produced by Ang II in vitro and inappropriately may raise questions regarding the significance of the local renin-angiotensin system in vivo in the pathological heart.
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grants HL-38132, HL-39902, HL-43023, and AG-15756 and by a Grant-in-Aid from the American Heart Association (No. 950321).
|
Footnotes |
|---|
Reprint requests to Piero Anversa, MD, Department of Medicine, Vosburgh Pavilion, Room 302, New York Medical College, Valhalla, NY 10595.
Received December 2, 1997; accepted March 25, 1998.
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