Download Preserved contractile function despite atrophic remodeling in

Am J Physiol Heart Circ Physiol
281: H1131–H1136, 2001.
Preserved contractile function despite atrophic
remodeling in unloaded rat hearts
DENISE C. WELSH,1 KONSTANTINA DIPLA,1 PATRICK H. MCNULTY,2 ANBIN MU,1
KAIE M. OJAMAA,2 IRWIN KLEIN,2 STEVEN R. HOUSER,1 AND KENNETH B. MARGULIES1
1
Cardiovascular Research Group, Temple University Medical Center,
Philadelphia, Pennsylvania 19140; and 2Division of Endocrinology,
North Shore University Hospital, Manhassett, New York 11030
Received 26 January 2001; accepted in final form 18 April 2001
LVAD support, a recent editorial comment by Soloff
(31) has raised the valid concern that reduced myocardial mass might limit or offset the potential benefits of
LVAD support. Such concerns are amplified by other
recent studies demonstrating that cardiac unloading
and atrophy produce activation of gene-expression patterns associated with fetal development and pathological hypertrophy (5).
In this context, it is reasonable to consider whether
myocardial unloading of normal myocardium with associated reductions of muscle mass necessarily result
in decreased pump performance. For the purpose of
this discussion, we use the term “atrophy” to denote
reductions in cardiac and/or myocyte size below normal
levels. On the basis of previous studies reporting that
atrophy of normal hearts is associated with changes in
gene expression resembling those observed with pathological hypertrophy (5, 20, 26), we hypothesized that
myocardial unloading would produce concordant decrements in both myocardial mass and function. To test
this hypothesis, we unloaded normal myocardium via
heterotopic transplantation (HT) in the rat, and performed morphological and functional analyses on isolated myocytes and force-mechanics studies on isolated
papillary muscles.
cardiac myocytes; heterotopic transplantation; papillary
muscles; morphometry
METHODS
have documented regression of pathological hypertrophy at the cellular level when hearts
with advanced dilated cardiomyopathy are supported
with a left ventricular assist device (LVAD) for several
weeks or months. To date, the changes in myocardial
phenotype induced by LVAD support have included
regression of abnormal protein expression associated
with pathological hypertrophy and failure (1, 2, 33),
improvements in distorted myocyte morphology (16,
34), and improvements in myocyte contractility and
Ca2⫹ handling (6, 7, 13). Despite these apparently
favorable responses to hemodynamic unloading via
Animals. Inbred male Lewis rats weighing 275–375 g were
used. All animals received humane care in compliance with
the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication
No. 85-23, Revised 1996). Approval for the studies performed
was obtained from the institutional animal care and use
committees at Temple University School of Medicine and the
North Shore University Hospital.
Surgical procedure. Heterotopic heart transplants were
performed using a surgical procedure that has been described
in detail previously (17, 22). In brief, rats were anesthetized
with an injection of pentobarbital sodium (50 mg/kg ip). The
chest was opened and the donor heart was exposed. Heparin
was administered and the vena cavae and pulmonary veins
were ligated. The heart was then excised and flushed with
Address for reprint requests and other correspondence: K. B.
Margulies, Cardiovascular Research Group, Rm. 805 MRB, Temple
Univ. School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140
(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
RECENT STUDIES
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0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
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Welsh, Denise C., Konstantina Dipla, Patrick H. McNulty, Anbin Mu, Kaie M. Ojamaa, Irwin Klein, Steven
R. Houser, and Kenneth B. Margulies. Preserved contractile function despite atrophic remodeling in unloaded rat
hearts. Am J Physiol Heart Circ Physiol 281: H1131–H1136,
2001.—The present study was designed to determine
whether myocardial atrophy is necessarily associated with
changes in cardiac contractility. Myocardial unloading of
normal hearts was produced via heterotopic transplantation
in rats. Contractions of isolated myocytes (1.2 mM Ca2⫹;
37°C) were assessed during field stimulation (0.5, 1.0, and 2.0
Hz), and papillary muscle contractions were assessed during
direct stimulation (2.0 mM Ca2⫹; 37°C; 0.5 Hz). Hemodynamic unloading was associated with a 41% decrease in
median myocyte volume and proportional decreases in myocyte length and width. Nevertheless, atrophic myocytes had
normal fractional shortening, time to peak contraction, and
relaxation times. Despite decreases in absolute maximal
force generation (Fmax), there were no differences in Fmax /
area in papillary muscles isolated from unloaded transplanted hearts. Therefore, atrophic remodeling after unloading is associated with intact contractile function in isolated
myocytes and papillary muscles when contractile indexes are
normalized to account for reductions in cell length and crosssectional area, respectively. Nevertheless, in the absence of
compensatory increases in contractile function, reductions in
myocardial mass will lead to impaired overall work capacity.
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PRESERVED CONTRACTILE FUNCTION IN ATROPHIC REMODELING
AJP-Heart Circ Physiol • VOL
Hz using a video edge-detection system (Crescent Electronics; Sandy, UT). Data were stored on a computer for later
analysis using Axotape software (Axon Instruments; Foster
City, CA). Resting myocyte length, fractional shortening
(change in length divided by resting length), time to peak
contraction, and time to 50% relaxation were determined in
8–12 cells from each heart. In a subset of 4–6 cells from each
heart, the frequency dependence of contraction was assessed
by increasing the stimulation frequency from 0.5 to 1.0 to 2.0
Hz. Steady-state fractional shortening was measured at each
stimulation frequency.
Papillary muscle function. A septal papillary muscle was
dissected from 10 normal Lewis rat hearts and 6 heterotopically transplanted donor hearts 2 wk after transplant surgery. Rats were anesthetized and a cardiectomy was performed as described above. The papillary muscles were
mounted horizontally at an arbitrary length between metal
clips on an isometric strain-gauge transducer in a Plexiglas
bath. The chamber was superfused with KHB containing 2.0
mM CaCl2 and equilibrated with 95% O2-5% CO2 at 37°C.
Differences in the CaCl2 concentrations used for isolated
myocytes and papillary muscles reflect established differences in the dose-response curves for Ca2⫹ between the two
preparations (3). The papillary muscle was stimulated with a
Grass square-wave stimulator (Grass Instruments; Braintree, MA) at a stimulation voltage 25% above threshold with
a 5-ms pulse width. After being mounted, each muscle was
equilibrated for 30 min while being stimulated at a frequency
of 2 stimulations/min. Muscle length was then gradually and
incrementally increased until the muscle attained the length
at which maximal developed force was achieved (Lmax). The
muscle was then stimulated twice per minute for the next 20
min at Lmax to confirm a stable (⬍10% variation during 20
min) developed force. Twitches from muscles achieving a
stable developed force were recorded on a two-channel paperchart recorder (Gould Instruments; Valley View, OH). For
experimental measurements, developed force and time to
50% relaxation were calculated for 5–10 consecutive contractions at a frequency of 0.5 Hz. The diameter of each muscle
was then measured in place with a calibrated microscope.
Cross-sectional area was calculated by assuming the muscle
had a cylindrical shape, and developed force was normalized
for the cross-sectional area of the muscle.
Statistical analysis. All data reported in the text and tables
are expressed as means ⫾ SE. Comparisons between heterotopically transplanted and control hearts (in both myocytes
and papillary muscles) were analyzed by unpaired Student’s
t-test using a commercially available software program (StatView version 4.1; Abacus Concepts; Berkeley, CA). A P value
⬍0.05 was considered statistically significant for all hypothesis testing.
RESULTS
Morphometric measurements. The results of organ
and cellular morphological analyses are presented in
Table 1. Compared with normal hearts from agematched rats, 2 wk of hemodynamic unloading via HT
was associated with a 23% decrease in the absolute
heart weight (P ⬍ 0.001) and a 26% decrease in the
heart weight-to-body weight ratio (P ⬍ 0.001). Of note,
paired comparisons of the weights of transplanted
hearts at the time of transplantation and at explantation 2 wk later demonstrated an average heart mass
decrease of 28 ⫾ 1% (P ⬍ 0.001).
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cold heparinized saline via a catheter in the ascending aorta.
The heart was kept in a cold, high-potassium, cardioplegic
buffer solution (30) until transplantation. Recipient rats
were anesthetized and prepared in the same manner as
donor rats. The abdomen was entered through a midline
incision and the abdominal aorta and inferior vena cava were
exposed and clamped. The donor heart was placed in the
right abdominal cavity and end-to-side anastomoses were
made between the donor’s aorta and the recipient’s abdominal aorta and between the donor’s pulmonary artery and the
recipient’s vena cava. Care was taken to keep the heart cold
throughout the surgical procedure, and the total duration of
cold ischemia ranged from 45 to 60 min for all allografts. This
procedure allows the intact, transplanted heart to be adequately perfused while remaining hemodynamically unloaded. Unoperated Lewis rats served as controls.
Myocyte isolation. Myocytes were isolated from seven normal Lewis rat hearts and six heterotopically transplanted
hearts 2 wk after heart transplantation. Rats were anesthetized with an injection of pentobarbital sodium (150 mg/kg
ip), and a cardiectomy was performed immediately. Each
heart was placed on a modified Langendorf perfusion apparatus and perfused with a nonrecirculating, nominally Ca2⫹free solution containing Krebs-Henseleit buffer (KHB) solution [in mM: 12.5 glucose, 5.4 KCl, 1 lactic acid, 1.2 MgSO4,
130 NaCl, 1.2 NaH2PO4, 25 NaHCO3, and 2 sodium pyruvate
(pH 7.4)] with 10 mM taurine until the coronaries were clear
of blood. The heart was then perfused for 10–15 min with a
recirculating digestion solution containing type II collagenase (180 U/ml) and 20 mM taurine. The heart was then
removed from the cannula and the right ventricular tissue
was removed before the digested left ventricle (LV) was
minced in the collagenase solution. The resulting cell suspension was filtered and centrifuged at 25 g. Isolated myocytes
were resuspended in a KHB solution containing 1% wt/vol
bovine albumin, 10 mM taurine, and 0.5 mM CaCl2. All
solutions were equilibrated with 95% O2-5% CO2. The temperature was kept at 37°C throughout the isolation procedure.
Myocyte morphometric analysis. Freshly isolated cells
were fixed in an isoosmotic solution of 1.5% glutaraldehyde in
0.06 M phosphate buffer. This fixative does not alter cell
volume or shape as previously shown by Gerdes and colleagues (12). For each fixed-cell suspension, average myocyte
morphological parameters were derived using a combination
of cell-volume measurement and isolated myocyte image
analysis as previously described (34). Median cell volume
was measured in an aliquot containing at least 10,000 cells
using a Coulter Channelyzer and a shape factor of 1.05 as
previously validated by Hurley (14). Image analysis was
performed using the NIH Image Analysis program (version
1.59) and a charge-coupled device camera, frame grabber,
and inverted microscope. A total of 60 myocytes were measured from each myocyte suspension, and cells were selected
based on visible striations and the absence of membrane
blebs or abnormal granularity. For each myocyte preparation, average myocyte cross-sectional area was calculated by
dividing median myocyte volume by mean myocyte length.
Isolated myocyte function. For analysis of basal myocyte
contractility, myocytes were placed in a chamber on the stage
of an inverted microscope. The chamber was superfused at
1–2 ml/min with Tyrode solution [containing, in mM: 150
NaCl, 5.4 KCl, 1 CaCl2, 1.2 MgCl2, 10 glucose, 2 pyruvate,
and 5 HEPES (pH 7.4)] at 37°C. Myocytes were chosen based
on the same criteria as for the morphometric analysis as well
as the absence of spontaneous contractions in 1 mM Ca2⫹.
Contractions were measured during field stimulation at 0.5
PRESERVED CONTRACTILE FUNCTION IN ATROPHIC REMODELING
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Table 1. Heart and myocyte morphology
Control Hearts
Number of animals
Body weight,† g
Heart weight, g
Heart weight/Body weight,†
g/kg
Median myocyte volume, ␮m3
Cell length, ␮m
Cell width, ␮m
Cell cross-sectional area, ␮m
7
315 ⫾ 12
1.12 ⫾ 0.05
3.58 ⫾ 0.12
31,115 ⫾ 1,571
140 ⫾ 12
33 ⫾ 4
225 ⫾ 12
Unloaded
Hearts
6
330 ⫾ 15
0.86 ⫾ 0.02*
2.63 ⫾ 0.09*
18,375 ⫾ 1,829
110 ⫾ 5*
26 ⫾ 3*
167 ⫾ 11*
Values are means ⫾ SE; * P ⬍ 0.05 vs. control hearts. † Donor rat
body weight in unloaded hearts.
Fig. 1. Representative steady-state contractions during field stimulation (at 0.5 Hz) of rat ventricular myocytes from heart unloaded
with 2 wk of heterotopic transplantation and nonunloaded controls.
Cells with equivalent resting cell length (130–133 ␮m) were chosen.
Fmax, or time to 50% relaxation in papillary muscles
isolated from unloaded transplanted hearts compared
with papillary muscles from control hearts. However,
absolute Fmax in muscles from transplanted hearts was
reduced compared with normal controls. As illustrated
in Fig. 3, this reduction was roughly proportionate to
the decrease in papillary muscle cross-sectional area
that was observed in unloaded hearts.
DISCUSSION
Cardiac unloading of normal myocardium and pathologically hypertrophied myocardium results in atrophic remodeling at both the organ and cellular levels
(1, 4, 8, 9, 17, 19, 27, 34). Data from the present studies
indicate that the normal myocardium of heterotopically transplanted rat hearts undergoes atrophic remodeling at the organ and cellular levels as would be
expected with the hemodynamic unloading created by
this model. This rapid remodeling is clearly shown by
the reduced heart weight-to-body weight ratio and the
decreased myocyte length, width, and volume in our
transplanted rat hearts. However, unlike previous
studies with this model, these studies demonstrate
Table 2. Myocyte and papillary muscle contractions
Control Hearts
Isolated myocyte studies
Number of cells
Fractional shortening, %
Time to peak contraction, ms
Time to 50% relaxation, ms
Isolated papillary muscle
studies
Number of muscles
Cross-sectional area, mm2
Fmax, mg
Fmax/area, g/mm2
Time to Fmax, ms
Time to 50% relaxation, ms
69
8.9 ⫾ 0.6
163 ⫾ 4
242 ⫾ 7
10
0.186 ⫾ 0.024
268 ⫾ 25
1.70 ⫾ 0.25
152 ⫾ 5
85 ⫾ 2
Unloaded Hearts
60
9.9 ⫾ 0.5
161 ⫾ 6
240 ⫾ 11
6
0.142 ⫾ 0.029*
184 ⫾ 53*
1.55 ⫾ 0.30
164 ⫾ 10
97 ⫾ 8
Values are means ⫾ SE. Fmax, maximal force generation. * P ⬍
0.05 vs. control hearts.
AJP-Heart Circ Physiol • VOL
Fig. 2. Stimulation frequency dependence of contraction in fieldstimulated rat ventricular myocytes. Average data from myocytes
unloaded with 2 wk of heterotopic transplantation (n ⫽ 30) and from
nonunloaded control myocytes (n ⫽ 34) are shown.
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At the cellular level, 2 wk of hemodynamic unloading
produced a 41% decrease in median myocyte volume.
This decrease in cell volume reflected both a 21% decrease in average myocyte length (P ⬍ 0.001) and a
26% decrease in average myocyte cross-sectional area
(P ⬍ 0.02). These cardiac and cellular morphological
findings demonstrate that 2 wk of myocardial unloading via HT is sufficient to produce marked organ and
cellular atrophy of the normal heart.
Function measurements. The contractile function
measurements for isolated myocytes and papillary
muscles from normal control hearts and heterotopically transplanted hearts are presented in Table 2. As
illustrated in Fig. 1 and summarized in Table 2, compared with normal cardiac myocytes, isolated myocytes
from unloaded hearts demonstrated no differences in
fractional shortening, time to peak contraction, or time
to 50% relaxation at 0.5 Hz. Recognizing that physiological differences might be more apparent during increased stimulation frequency, we examined the fractional shortening of 34 myocytes from control hearts
and 30 myocytes from unloaded hearts at stimulation
frequencies of 0.5, 1.0, and 2.0 Hz. As shown in Fig. 2,
equivalent fractional shortening of control and unloaded myocytes was also observed during progressive
increases in stimulation frequency.
In isolated papillary muscles, there were no differences in the peak developed force (Fmax)/area, time to
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PRESERVED CONTRACTILE FUNCTION IN ATROPHIC REMODELING
Fig. 3. Regression plot demonstrating the relationship between the
cross-sectional area and maximally developed force (Fmax) in isolated
papillary muscle preparations from rat hearts with (E) and without (F)
antecedent hemodynamic unloading by heterotopic transplantation.
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intact contractile function at both the cellular and
muscular levels when myocyte shortening is normalized to resting cell length and when papillary muscle
force generation is normalized to cross-sectional area.
These findings of preserved function using normalized
indexes of contractility suggest that atrophic remodeling does not adversely affect sarcomere function under
basal conditions. Nevertheless, the striking reduction
in absolute Fmax as a result of atrophic remodeling does
demonstrate that a net loss of sarcomeres and myocardial mass will reduce myocardial work capacity even
when sarcomere function is preserved.
Similar to previous studies of in vivo cardiac unloading of the normal heart (4, 8, 9, 17–19), the present
studies demonstrated morphological atrophy at the
organ and tissue levels. Specifically, we observed a 23%
reduction in heart weight and a 27% reduction in heart
weight-to-body weight ratio at 2 wk after HT. At the
cellular level, we observed that average myocyte volume in unloaded hearts was 41% smaller than in
normally loaded control hearts. In addition to demonstrating that organ atrophy is a reflection of cellular
atrophy, our cellular morphometrics also demonstrate
for the first time that unloading of the normal heart
produces balanced reductions in both cellular length
and cross-sectional area consistent with reductions in
both series and parallel sarcomeres. These proportionate decreases in length and cross-sectional area after
unloading of the normal heart are similar to the balanced 20% decreases in myocyte length and width
observed by Zafeiridis and colleagues (34) after mechanical unloading with a LVAD in severely failing
human hearts (34). However, the present finding contrasts with the disproportionately large (14%) decreases in myocyte cross-sectional area compared with
relatively smaller (6%) decreases in myocyte length
observed by Gerdes and colleagues (11) after closing a
chronic aortocaval fistula in rats. These distinctions
may reflect the more complete reductions in both ventricular preload and afterload produced by HT and
mechanical circulatory support.
The present studies evaluated isolated myocyte contractility in the atrophied heart. These in vitro studies
revealed fractional shortening and relaxation re-
sponses in myocytes from atrophied hearts that did not
differ from those observed in nonatrophied normal
controls. By design, functional studies using isolated
myocytes assess myocyte contraction and relaxation in
the absence of preload or afterload. Our findings demonstrate that load-independent shortening is normal in
myocytes with morphological atrophy. As is conventional in such studies, we measured fractional shortening to provide an index of sarcomere shortening.
Thus our findings indicate that basic sarcomere function is intact despite morphological atrophy. Because
changes in the rates of contraction and relaxation often
precede changes in the magnitude of shortening, our
findings of normal times to peak contraction and 50%
relaxation in unloaded myocytes further attest to preservation of contractile function. Finally, equivalent responses to increasing stimulation frequency also suggest intact contractile function in unloaded myocytes.
Beyond the normal cellular contractility that we observed in atrophic myocytes, a recent study by Ritter
and colleagues (29) reported supranormal contractility
and Ca2⫹ transients in mouse myocytes 5 days after
HT. Although distinctions between the present findings and this previous report could reflect species differences or the differences in the duration of unloading,
the observations nevertheless support the contention
that morphological atrophy need not be associated with
impairment of sarcomere function. On the other hand,
preliminary observations by Ito and co-workers (15)
suggest a slowing of contraction kinetics and decreased
contractile reserve in rat myocytes after 5 wk of hemodynamic unloading. Together with the present studies,
the studies of Ritter and colleagues (29) and Ito and
co-workers (15) suggest possible time-dependent
changes in myocyte contractility and contractile reserve in the setting of morphological atrophy with
initial increases and subsequent decreases.
Despite morphological atrophy evidenced by reduced
cross-sectional area, isolated papillary muscles from
unloaded hearts exhibited isometric force generation
which was equivalent to that observed in normal
hearts when Fmax was normalized by cross-sectional
area. Because muscle cross-sectional area reflects the
number of sarcomeres in parallel, our findings in papillary muscles suggest that basic sarcomere function is
intact despite morphological atrophy. In this regard,
our findings in isolated papillary muscles are analogous to our findings in isolated myocytes. As in the
myocyte studies, the normal time to Fmax and the
normal time to 50% relaxation in atrophied papillary
muscles further attest to intact contractile performance. Our finding of normal Fmax/area in atrophied
papillary muscles under basal conditions is consistent
with previous studies by Korecky and co-workers (21)
who studied rat isografts 8 wk after HT. A relevant
clinical example of the same phenomenon are studies
in which LV ejection fraction remained normal while
parallel reductions in LV afterload and LV mass were
observed in women with anorexia nervosa (32). As in
the present study, these previous findings demonstrate
that reductions in cardiac workload trigger reductions
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AJP-Heart Circ Physiol • VOL
also possible that previously reported changes in myosin isoform and glucose metabolism could affect responses to physiological stress without altering basal
contractile responses as assessed in the present studies. Ultimately, new studies will be required to determine the conditions under which molecular changes
associated with myocardial atrophy are of functional
significance.
The present studies show that despite morphological
atrophy, myocardial unloading via HT is associated
with intact contractile function in isolated myocytes
and papillary muscles when contractile indexes are
normalized to account for differences in cell length and
cross-sectional area, respectively. Based on our findings of reduced absolute Fmax, it is likely that previously reported reductions in global cardiac function
after HT (8, 9) could simply be a reflection of the
reduced cardiac mass. These observations have potential implications for recent observations in diseased
human hearts unloaded via mechanical circulatory
support. On one hand, improved fractional shortening
observed in isolated myocytes (6) should improve overall cardiac contractile performance. On the other hand,
the findings in the present study suggest that reductions in organ and cellular mass after mechanical circulatory support (1, 34) may tend to offset gains resulting from improvements in contractile function.
Consideration of such potential trade-offs involved in
reverse remodeling during mechanical cardiac support
may help optimize contractile improvements in failing
hearts at both the organ and cellular levels.
This research was supported by National Institutes of Health
Grants HL-03560 and AG-17022 to K. B. Margulies and HL-39201
and HL-61495 to S. R. Houser.
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