23
Alterations in Cardiac Gene Expression During
the Transition From Stable Hypertrophy
to Heart Failure
Marked Upregulation of Genes Encoding
Extracellular Matrix Components
Marvin
0.
Boluyt, Lydia O'Neill, Andrea L. Meredith, Oscar H.L. Bing, Wesley W. Brooks,
Chester H. Conrad, Michael T. Crow, Edward G. Lakatta
Abstract The failing heart is characterized by impaired
cardiac muscle function and increased interstitial fibrosis. Our
purpose was to determine whether the functional impairment of
the failing heart is associated with changes in levels of mRNA
encoding proteins that modulate parameters of contraction and
relaxation and whether the increased fibrosis observed in the
failing heart is related to elevated expression of genes encoding
extracellular matrix components. We studied hearts of 18- to
24-month-old spontaneously hypertensive rats with signs and
symptoms of heart failure (SHR-F) or without evidence of
failure (SHR-NF) and of age-matched normotensive WistarKyoto (WKY) rats. Compared with WKY rats, SHR-NF exhibited left ventricular (LV) hypertrophy (2.2-fold) and right
ventricular (RV) hypertrophy (1.5-fold), whereas SHR-F were
characterized by comparable LV hypertrophy (2.1-fold) and
augmented RV hypertrophy (2.4-fold; all P<.01). Total RNA
was isolated from ventricles and subjected to Northern blot
analysis. In SHR-F hearts, the level of a-myosin heavy chain
mRNA was decreased in both ventricles to 1/3 and '/s of the
SHR-NF and WKY values, respectively (both P< .01). Levels of
,l-myosin heavy chain, a-cardiac actin, and myosin light chain-2
mRNAs were not significantly altered in hearts of SHR-NF or
SHR-F. Levels of a-skeletal actin were twofold greater in
SHR-NF hearts compared with WKY hearts and were intermediate in SHR-F hearts. Levels of atrial natriuretic factor (ANF)
mRNA were elevated threefold in the LV of SHR-NF (P<.05)
but were not significantly increased in the RV of SHR-NF
compared with WKY rats. During the transition to failure
(SHR-F versus SHR-NF), ANF mRNA levels increased an
additional 1.6-fold in the LV and were elevated 4.7-fold in the
RV (both P<.05). Levels of sarcoplasmic reticulum Ca2ATPase (SRCA) mRNA were maintained in the LV of hypertensive and failing hearts at levels not significantly different
from WKY values. In contrast, the level of RV SRCA mRNA
was 24% less in SHR-NF compared with WKY rats, and during
the transition to failure, this difference was not significantly
exacerbated (29% less than the WKY value). The levels of
fibronectin and pro-al(I) and pro-al(III) collagen mRNAs
were not significantly elevated in either ventricle of the
SHR-NF group but were fourfold to fivefold higher in both
ventricles of SHR-F (all P<.05). The increase in fibronectin
gene expression was at least partially explained by an elevation
in the level of the EIILA-containing isoform, an alternatively
spliced variant expressed during wound healing and pressure
overload hypertrophy. Transforming growth factor-fl (TGF-fl1)
mRNA abundance was not elevated in ventricles of SHR-NF
but increased 1.3-fold in the LV and twofold in the RV during
the transition to heart failure compared with SHR-NF values
(both P<.05). The decrease in a-myosin heavy chain mRNA
levels in SHR-F hearts represents a pretranslational basis for
the slowed contraction previously observed in cardiac muscle
from these hearts. The survey of specific contractile protein
mRNAs provides no evidence of a downregulation of these
genes during the transition to heart failure. The increase in
fibronectin and collagen mRNA levels suggests that the previously observed increase in interstitial fibrosis in cardiac muscles
of failing hearts is regulated at the level of gene expression. The
increase in abundance of TGF-,f1 mRNA in conjunction with
the upregulation of fibronectin and collagen genes suggests that
activation of TGF-fl1 gene expression may be a mechanism
initiating interstitial fibrosis during the transition from stable
hypertrophy to failure. (Circ Res. 1994;75:23-32.)
Key Words * heart failure * spontaneously hypertensive
rats * myosin heavy chain * myosin light chain * a-actin
* sarcoplasmic reticulum Ca`2-ATPase * fibronectin .
collagen * transforming growth factor-fl
linically diagnosed heart failure is a progressive
disease with a poor prognosis.' Heart failure
with hypertension.2 The hypertrophied heart that subsequently develops failure is characterized by altered
hemodynamics,3 impaired cardiac muscle function,4 6
and increased fibrosis.7,8 Although cardiac hypertrophy
is commonly associated with heart failure,' it is widely
recognized as an adaptive response that normalizes wall
stress and compensates for an increased load.9 When
the load is chronically elevated for an extended period
of time, such as in chronic hypertension, compensated
hypertrophy may progress to pump failure. One difficulty in studying the development of heart failure has
been the lack of a well-characterized animal model that
C
has many etiologies but is commonly associated
Received July 19, 1993; accepted March 1, 1994.
From the Laboratory of Cardiovascular Science, Gerontology
Research Center, National Institutes of Health, Baltimore, Md;
and the Department of Medicine, Boston Veterans Affairs Medical Center (Mass).
Correspondence to Marvin 0. Boluyt, PhD, Laboratory of
Cardiovascular Science, Gerontology Research Center, Room
3-E-10, 4940 Eastern Ave, Baltimore, MD 21224.
© 1994 American Heart Association, Inc.
24
Circulation Research Vol 75, No 1 July 1994
clearly exhibits a transition from compensated hypertrophy to failure. The spontaneously hypertensive rat
(SHR) has been found to develop impaired myocardial function after a period of stable hypertrophy.
Trippodo and Frohlich10 have summarized the evidence supporting the use of the SHR as a model of
human heart disease and heart failure. The intrinsic
myocardial properties of these animals have been well
characterized in terms of functional and morphological features.611-'13
Although the mechanism(s) that contributes to the
transition from compensated cardiac hypertrophy to
heart failure has remained obscure, several hypotheses
have been formulated. There is evidence to suggest that
a loss of contractile protein from the myocardium may
contribute to the impaired function observed in cardiac
muscle from failing human hearts.14 Myocyte dropout
due to myocardial cell death and hypertrophy of the
remaining viable myocytes has been proposed to account for impaired function and failure during aging
and hypertension.15-17 Since left ventricular (LV) mass
of SHR increases progressively with age and does not
diminish during failure as myocytes are lost, additional
nascent hypertrophy of surviving myocytes may occur.
Increased expression of muscle-specific genes such as
a-skeletal actin, which is transiently upregulated during
hypertrophy,18 would constitute evidence in support of
this hypothesis. Hypertrophied and, particularly, failing
hearts are characterized by an accumulation of extracellular matrix proteins and a corresponding increase in
cardiac muscle stiffness.78 Since fibronectin and collagen types I and III are major components of the
interstitial fibrillar network, it has been hypothesized
that upregulation of genes encoding these components
by fibroblasts accounts, in part, for the increase in
fibrosis observed during the transition to failure and
contributes to the decline in contractile performance.8
Of particular interest is the fibronectin mRNA-splicing
variant encoding the EIIIA segment. This isoform is
expressed during wound healing,19 embryogenesis,20
and pressure-overload hypertrophy.2'
The purpose of this investigation was to determine
whether there are alterations in expression of genes that
code for proteins potentially involved in functional
impairment observed in the myocardium of SHR with
signs and symptoms of heart failure (SHR-F). To differentiate alterations in gene expression associated with
hypertrophy from those affiliated with failure, we compared relative mRNA levels in hearts of three agematched groups: normotensive Wistar-Kyoto (WKY)
rats, hypertensive rats without failure (SHR-NF), and
SHR-F. Since the increased hemodynamic load imposed on the heart exerts its primary effect on the LV
but may impact the right ventricle (RV) significantly
during failure, we measured relative mRNA levels in
the RV and LV. Specifically, we studied the mRNA
levels of several contractile proteins, including
and
,B-myosin heavy chain (a- and ,B-MHC, respectively),
a-cardiac actin, and myosin light chain-2 (MLC2) to
investigate the possibility that a generalized paucity of
contractile protein mRNAs could contribute to the
observed contractile deficits and to determine whether
a pretranslational basis exists for the myosin isoform
shift. The levels of a-skeletal actin and and ,B-MHC
a-
a-
mRNA were studied as markers of cardiac hypertrophy
to determine whether there was any basis for nascent
hypertrophy in hearts with chronic hypertension and
failure. Atrial natriuretic factor (ANF) gene expression
was studied because it has been considered to be one of
several criteria indicating the initiation of a pathological
response.2 Since previous studies have shown a decrease in sarcoplasmic reticulum Ca2+-ATPase (SRCA)
gene expression in hypertrophied heart23 and in hearts
of human patients with heart failure,24 we investigated
the levels of SRCA in hearts of rats undergoing a
transition from stable hypertrophy to failure. To determine whether increased expression of fibroblast-specific
genes underlies the increase in fibrosis previously observed in failing hearts, we measured relative levels of
fibronectin and collagen mRNAs. Since the elaboration
of extracellular matrix by fibroblasts is influenced by
transforming growth factor-n (TGF-13)25 and since pressure-overload cardiac hypertrophy is associated with an
increase in TGF-,1 ,21 we also studied the levels of
TGF-f,1 mRNA.
Materials and Methods
Animals
Male retired breeder SHR and WKY rats (6 to 9 months
old) were purchased from Taconic Farms, Inc, and boarded in
the animal facility at the Boston Department of Veterans
Affairs Medical Center until the time of study (18 to 24 months
of age). Beginning at 18 months of age, rats were monitored
for evidence of rapid labored respiration and studied within 1
week of identification.6 At autopsy, animals were examined for
pleural/pericardial effusions, atrial thrombi, and RV hypertrophy. Several SHR with no findings other than RV hypertrophy
were excluded from the study. Individual chambers were
quickly dissected, and chamber weights were recorded. LV,
lung, and liver weights were determined before and after
drying sections of these tissues in an oven at 60WC.6 Cardiac
tissue samples were frozen within minutes of the death of the
animal and stored in liquid nitrogen until studied.
RNA Analysis
RNA was isolated from LV and RV by the method of
Chomczynski and Sacchi.26 Ten micrograms of total RNA was
size-fractionated by electrophoresis through 1% agarose gels,
transferred electrophoretically at 5 V/cm to a nylon (Duralon)
membrane, and hybridized with "2P-radiolabeled probes overnight at 63.5°C for cDNA probes and 60°C for oligonucleotide
probes.27 Hybridization intensity was quantified in disintegrations per minute directly from blots with a Betascope 603
(Betagen Corp). This apparatus contains an electronic sensor
that responds to beta radiation from all areas of the blot
directly with an efficiency of at least 15%. Signals visualized on
computer screen were identified by position relative to 18S and
28S rRNA migration, delineated by 9x9-mm rectangles, and
quantified. Each blot was subsequently stripped and reprobed.
The signal from each sample was normalized to the signal
obtained with a probe specific for the 18S ribosomal RNA.
Probes
Complementary DNA probes were synthesized from a
template by the random-prime method.2829 The templates
for the a-cardiac actin, a-skeletal actin, and ANF probes
were 180- to 700-bp cDNA sequences, which were obtained
by polymerase chain reaction (PCR) with primers complementary to the published sequences for rat mRNA.30-32 The
PCR-generated probe sequences were verified by DNA
sequence analysis. The fibronectin probe was a 2.2-kb prod-
Boluy et al Gene Expression in the Failing Heart
uct of an EcoRI digest of the 3-kb pRCabFN2 clone generously supplied by K. Boheler. This probe does not distinguish
between alternatively spliced fibronectin mRNA variants. To
identify EIIIA+ fibronectin mRNA, an oligonucleotide specific for this segment was constructed with the following
sequence: 5YCCACCGTGCAAGGCAACCACACTGACTGTGTACTCAG3' . The probes for pro-al(I) and proal(III) collagen were cDNAs kindly provided by M.-L.
Chu.34-35 The probe for TGF-lI was generously provided by
M. Sporn.36 The probes for c-MHC, l3-MHC, and the 18S
ribosomal RNA were end-labeled synthetic oligonucleotides.37 The probe for cardiac SRCA was also a synthetic
end-labeled oligonucleotide complementary to the terminal
33 bases of the coding portion of the rat gene38 with the
following sequence: 5' CTC CAG TAT TGC AGG CTC
25
TABLE 1. Biometric Data of Wistar-Kyoto Rats and Rats
Without and With Heart Failure
WKY
(n=12)
20.2+0.3
Age, mo
BW, g
LVW, g
RVW, g
LVW/BW, mg/g
SHR-NF
(n=12)
19.6+0.4
SHR-F
(n=16)
19.4+0.6
Results
372+20*
382+12*
1 .40 + 0.08
1.38 +0.05
0.26±0.02*
0.44+0.02*t
3.78+0.13*
3.66±0.13*
RVW/BW, mg/g
0.70±0.03*
1.14+0.04*t
LV W/D
4.70+0.03
4.85+0.04*t
Liver W/D
3.32±0.03
3.52+t0.07*t
Lung W/D
5.62±.020
5.48+0.06
WKY indicates Wistar-Kyoto rats; SHR-NF, spontaneously
hypertensive rats (SHR) without heart failure; SHR-F, SHR with
heart failure; BW, body weight; LVW, left ventricular (LV) wet
weight; RVW, right ventricular (RV) wet weight; and W/D, wet
weight-to-dry weight ratios (g/g). Values are mean-SEM.
*P<.01 vs WKY; tP<.01 vs SHR-NF.
Biometric and Clinical/Pathological Data
Body weight of both SHR-NF and SHR-F was '==50%
of the age-matched WKY rats for this cohort of rats
(Table 1). LV weight was similar for all three groups.
The LV weight-to-body weight ratio in SHR-NF and
SHR-F was more than twofold greater than the WKY
value. There was no difference in the magnitude of LV
hypertrophy in SHR-F compared with SHR-NF. The
RV weight-to-body weight ratio was elevated 49% in
SHR-NF compared with WKY rats and was an additional 63% greater in SHR-F compared with SHR-NF.
Thus, the transition from stable hypertrophy to heart
failure in SHR was characterized by a marked augmentation of hypertrophy in the RV but no additional LV
hypertrophy.
Among the SHR-F, 100% had visibly detectable
fibrosis, 69% exhibited pleural effusion, 81% were iden-
tified with atrial thrombi, and 94% had rapid/labored
respiration. The LV wet weight-to-dry weight ratio
was slightly elevated in SHR-F compared with WKY
and SHR-NF values (Table 1). The liver wet weightto-dry weight ratio was significantly elevated in the
SHR-F group, suggesting elevated right-side filling pressures. Lung wet weight-to-dry weight ratios were not
different among the groups, indicating that the lung may
be protected against fluid accumulation during elevated
hemodynamic pressures, as suggested by Erdmann et
al.39 Findings consistent with the presence of heart
failure are consonant with previous studies of the
SHR.6,11-13
Contractile Protein mRNA Levels
Previous studies of contractile function in the myocardium of these rats indicated impaired muscle func-
CAG GTA GTT TCG GGC 3.
Statistical Analysis
One-way ANOVA was used to evaluate differences among
the three groups, and post hoc comparisons between groups
were performed by Tukey's procedure. Differences were considered significant at P<.05.
721-+-26
1.23-+0.04
0.34+0.01
1.72±0.03
0.47+0.01
4.57±0.03
3.26+0.03
5.77+0.27
LV
a-MHC-
flpe*neem..nc_m.
18SLane
1
l
2 3 4 5 6 7 8 9 10 11 1213 14 1L5 16
WKY
SHR-Fl SHR-NF-
14
T
LV
12
0U
10
C)
8
MM**
%*"m
RV
r
6t
* *
4
FIG 1. a-Myosin heavy chain (a-MHC) mRNA levels in
hearts of Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) without heart failure (SHR-NF),
and SHR with heart failure (SHR- F). Top, Autoradiograph of Northern blot with total RNA from left ventridcles (LVs) hybridized with an oligonucleotide probe
specific for a-MHC. Signals were quantified on a
Betascope 603, followed by autoradiography. Membranes were subsequently stripped and hybridized
with an oligonucleotide probe for the 18S ribosomal
RNA. Bottom, Bar graphs showing Northern blot data
for LV and right ventricle (RV). Differences in loading
were corrected by normalizing the signal to that obtained for 18S ribosomal RNA from the same lane.
Values are mean-+-SEM for 8 to 12 samples per group.
Values represent arbitrary units; the a-MHC value for
the LV of the SHR-F group was arbitrarily set at 1.00,
and the remaining values were adjusted correspondingly. **P<.01 vs SHR-NF by ANOVA and Tukey's
procedure.
2
SHR-F
SHR-F
26
Circulation Research Vol 75 No 1 July 1994
LV
8-MHC-
on
18S-
Lane
s-
_~
0
a"
mt
em-
IN
t
2 3 4 5 6
WKY-J I
FIG 2. p-Myosin heavy chain (,B-MHC) mRNA levels in
hearts of Wistar Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) without heart failure (SHR-NF), and
SHR with heart failure (SHR-F). Top, Northern blot analysis with an oligonucleotide probe specific for f-MHC.
Bottom, Bar graphs showing Northern blot data. Differences in loading were corrected by normalizing the signal
to that obtained for 18S ribosomal RNA from the same
lane. Values are mean+SEM for 8 to 12 samples per
gop
7
8
9 10 11 1213 14
SHR-W-F
15
16
-SHR-F---l-
3-
RV
LV
U)
CD
ua
group. Values represent arbitrary units; the p-MHC value
for the left ventricle (LV) of the WKY group was arbitrarily
set at 1.00, and the remaining values were adjusted
correspondingly. RV indicates right ventricle.
2
-T-
I
r
1-
o
SHR-NF
SHR-F
WKY
SHR-NF
tion and an MHC shift in favor of the f3-isoform during
the transition from stable hypertrophy to failure.6
In
the present study, heart failure was associated with a
decrease in a-MHC mRNA in both ventricles. Compared with the WKY value, the LV level of a-MHC
mRNA was not significantly different in the ventricles
of SHR-NF but decreased in the LV of SHR-F to less
than one third of the SHR-NF value (Fig 1). Although
a-MHC mRNA levels were :"2.5 -fold higher in the RV
than in the LV of all groups (P<.05 by paired t test), the
relative loss in the SHR-NF was similar (Fig 1). Levels
of ,B-MHC were not significantly upregulated but remained comparable to the WKY values in ventricles of
both SHR-F and SHR-NF and were comparable in both
the LV and RV (Fig 2). Given the interanimal variability in f3-MHC mRNA levels and the size of the sample
reported here (n=10 to 12 per group), an increase of
less than twofold would be statistically undetectable at
the 95% confidence level (power analysis).41n The LV
levels of a-cardiac actin and MLC2 mRNAs were not
significantly altered during hypertension or failure (Table 2). It should be noted that given the variability and
sample size (n=5 or 6 per group), the smallest difference detectable at the 95% confidence level would be a
37% decrease for a-cardiac actin and a 55% decrease
for MLC2. LV levels of a-skeletal actin mRNA were
twofold greater in hearts of the SHR-NF compared with
the WKY group and were intermediate in the SHR-F
group (Table 2).
ANF mRNA Levels
Elevated levels of ANF mRNA were present in both
ventricles of failing hearts. Steady-state levels of ANF
mRNA were elevated threefold in the LV of SHR-NF
compared with WKY rats but were not significantly
increased in the RV (Table 2). In SHR-F, ANF mRNA
levels were increased an additional 1.6-fold in the LV
relative to SHR-NF levels and were incremented 4.7fold in the RV. ANF mRNA abundance was 2.5- to
SHR-F
9-fold greater in the LV compared with the RV
(P<.05).
SRCA mRNA Levels
Levels of SRCA mRNA were maintained in the LV
of hypertensive and failing hearts at levels not significantly different from WKY values (Fig 3). Given the
sample size of five or six per group and the variability
observed, the smallest change detectable in the LV at
the 95% confidence level would be a 40% decrease. In
contrast, the level of RV SRCA mRNA was 24% less in
SHR-NF compared with WKY rats, and during the
transition to failure, this decrease was not significantly
exacerbated (29% less than the WKY value).
TABLE 2. Atrial Natriuretic Factor, a-Cardiac Actin,
a-Skeletal Actin, and Myosin Light Chain-2 Gene
Expression in Hearts of Wistar-Kyoto Rats and
Spontaneously Hypertensive Rats Without and
With Heart Failure
WKY
SHR-NF
SHR-F
LV
RV
1.00+0.34
0.21 +.05
3.21+0.45*
0.35+0.06
5.19±0.39*t
LV
1.00+0.10
2.26+0.26*
1.63+0.21
LV
1.00+0.15
0.81±0.12
0.94±0.10
ANF
2.01 0.43*t
SK
CA
MLC2
LV
1.00 +0.26
1.02 +0.24
0.91±0.17
WRY indicates Wistar Kyoto rats; SHR-NF, spontaneously
hypertensive rats (SHR) without heart failure; SHR-F, SHR with
heart failure; ANF, atrial natriuretic factor; LV, left ventricle; RV,
right ventricle; SK, a-skeletal actin; CA, a-cardiac actin; and
MLC2, myosin light chain-2. Values are mean-+-SEM for 5 to 14
samples per group.
*PC.<5 vs WKY; tP<.05 vs SHR-NF.
Boluyt et al Gene Expression in the Failing Heart
LV
27
EIIIAA
SRCA
Ufl"P
1 8 S-
Lane
18SLane
RV
1.4
WKY
SHR-NF
SHR-F
WKV
Levels of sarcoplasmic reticulum Ca2
7 8 9 10 11 1213 14 15
SHR-FW L A F
SHR-NF-FIG 5. Autoradiogram of Northern blot analysis showing levels
of EIIIA+ fibronectin mRNA in the left ventricle of Wistar-Kyoto
(WKY) rats, spontaneously hypertensive rats (SHR) without heart
failure (SHR-NF), and SHR with heart failure (SHR-F). The blot
was probed with an oligonucleotide specific for the EIIIA segment of fibronectin mRNA. The blot was subsequently stripped
and probed for the 1 8S ribosomal RNA, indicating approximately
equivalent loading of lanes. Betascope analysis indicates a
threefold to fivefold increase in levels of EIIIA+ fibronectin in
SHR-F compared with SHR-NF and WKY rats. However, quantification must be interpreted with caution, since the WKY and
SHR-NF values were barely distinguishable from the background signal. L indicates 20 gg of rat liver RNA (positive for
fibronectin but negative for EIIIA+ fibronectin33); A, 20 gg of
atrial RNA (positive for EIIIA+ fibronectin41); and F, 10 gg of
neonatal rat cardiac fibroblast RNA.
1 2 3 4 5 6 7 8 9 10 11 1213 14
WKYSHR-F
SHR-NF-^J
FIG 3.
1 2 3 4 5 6
L-WKYJ I
fhlf
SHR-NF
SHR-F
-ATPase
(SRCA)
mRNA in the ventricles of Wistar-Kyoto (WKY) rats, spontaneously
hypertensive rats (SHR) without heart failure (SHR-NF), and SHR
with heart failure
analysis for the
(SHR-F).
Top, Autoradiogram of Northern blot
left ventricle
(LV).
Blots were
probed
with
an
EIIIA-containing isoform (Fig 5). Pro-al(I) and proal(IIJ) collagen mRNA levels in the LV and RV of
oligonucleotide specific for the cardiac SRCA. Bottom, Bar graphs
showing
mean+SEM
of five
or
six
determinations
from
each
SHR-F were also elevated threefold to fourfold over
WKY and SHR-NF values (Figs 6 and 7). Although the
levels of pro-al (I) and pro-al(III) collagen mRNAs did
not differ between the LV and RV, fibronectin mRNA
abundance was twofold to fourfold greater in the LV
than in the RV (P<.05). Levels of TGF-f31 mRNA did
not differ between SHR-NF and WKY rats in either
ventricle. However, there was a small but significant
increase in TGF-f3 mRNA levels in both ventricles of
SHR-F (Fig 8). Despite a nearly fourfold higher level of
TGF-P3 gene expression in the LV compared with the
RV (P<.05), the magnitude of the increment exhibited
during the transition to heart failure was similar in both
ventricles.
group for the LV and seven determinations per group for the right
ventricle (RV) by Northern blot analysis. Values represent arbitrary
units; the WKY values were set at 1 .00 for each ventricle, and the
remaining values were adjusted accordingly.
*Pc.05 vs
WKY rats
by ANOVA and Tukey's procedure.
Extracellular Matrix-Associated mRNA Levels
Levels
of
fibronectin
and
collagen
mRNAs
were
increased in both ventricles of failing hearts. The levels
of fibronectin mRNA were not significantly elevated
either ventricle of the
SHIR-NE
in
group but were fourfold
to fivefold higher in both ventricles of SHR-F (Fig 4).
The increase in fibronectin gene expression was at least
partially explained by an elevation in the level of the
LV
FN-
S.
18S- *
W
lsr
T
LV
i
z
E
RV
[
10
0
c
io
FIG 4. Levels of fibronectin mRNA in ventricles of WistarKyoto (WKY) rats, spontaneously hypertensive rats (SHR)
without heart failure (SHR-NF), and SHR with heart failure
(SHR-F). Top, Autoradiogram of Northern blot analysis.
Blots were probed with a cDNA probe for rat fibronectin.
Bottom, Bar graphs showing data from Northern blot.
Values are mean+SEM of 8 to 11 samples per group.
Values represent arbitrary units; the WKY left ventricular
(LV) value was set at 1.00, and the remaining values were
adjusted accordingly. RV indicates right ventricle.
**P<.05 vs SHR-NF by ANOVA and Tukey's procedure.
wW t
w
1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16
WKY- SHR-NF
SHR F
Lane
0
flflWflW
*
5
U.
WKY
SKR-NF
SHR-F
WKY
SHR-NF
*
28
Circulation Research
Vol 75, No 1 July 1994
LV
C1-
18S.
meOn
.
-4
*b*nfl
e
*
t1
' 00
qo
1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16:17 18
x
SHR-W---WKY
SHR-Fl
Lane
$
LV
z
E
E
RV
-F
4
* *
S
FIG 6. Levels of pro-al (1) collagen mRNA in ventricles of
Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats
(SHR) without heart failure (SHR-NF), and SHR with heart
failure (SHR-F). Top, Autoradiogram of Northern blot
analysis. Blots were probed with a cDNA probe for rat
fibronectin. Bottom, Bar graphs showing data from Northern blot. Values are mean-+-SEM of 6 to 13 samples per
group. Values represent arbitrary units; the WKY left
ventricular (LV) value was set at 1.00, and the remaining
values were adjusted accordingly. RV indicates right
ventricle. **P<.05 vs SHR-NF by ANOVA and Tukey's
procedure.
c
2
S
I
U
t
Di
WKY
SHR-NF
nnm-r
Discussion
mRNA levels but no increase in levels of ca-skeletal
actin mRNA (which was increased in SHR-NF compared with WKY rats). There was no significant decrease in SRCA mRNA levels in either ventricle during
the transition to failure. The most striking characteristic
distinguishing failing hearts from nonfailing hearts of
hypertensive rats was a threefold to fivefold increase in
fibronectin and collagen mRNAs in both ventricles. The
marked increase in mRNAs encoding extracellular matrix component proteins in failing hearts was accompanied by a small but significant elevation in TGE-j3,
mRNA, suggesting that increased expression of the
TGF-f1 gene may be a mechanism contributing to the
elaboration of extracellular matrix and deterioration of
function that characterize the failing heart.
The transition from compensated hypertrophy to
heart failure in the SHR is associated with augmented
RV hypertrophy, no additional LV hypertrophy, impaired LV muscle function,6" 12 and marked alterations
in the expression of both myocyte-specific and fibroblast-specific genes. The present results document a
significant loss of a-MHC mRNA from both ventricles
of failing hearts, with no significant change in 3-MHC
mRNA levels. There was no evidence of a generalized
decrease in LV contractile protein mRNA levels during
failure, because the abundance of a-cardiac actin and
MLC2 mRNA was maintained at values similar to those
of nonfailing and nonhypertensive control hearts. Failing hearts exhibited a biventricular increase in ANF
LV
0
C3-
18S
-
Lane
0
1
n tl
7 8 9 10 11 12 13 14 1S 16 17 18
l SHR-F- -J s
SHR-NF--
1 2 3 4 5 6
---WKY--l l
* *
6
LV
(o
RV
T
5
.4
z
E
*
*
4
3
0
c
ci
2
OL
WKY
SHR-NF
SHR-F
WKY
SH
FIG 7. Levels of pro-al(lIl) collagen mRNA in ventricles
of Wistar-Kyoto (WKY) rats, spontaneously hypertensive
rats (SHR) without heart failure (SHR-NF), and SHR with
heart failure (SHR-F). Top, Autoradiogram of Northern blot
analysis. Blots were probed with a cDNA probe for rat
fibronectin. Bottom, Bar graphs showing data from Northern blot. Values are mean±SEM of four to seven samples
per group. Values represent arbitrary units; the WKY left
ventricular (LV) value was set at 1.00, and the remaining
values were adjusted accordingly. RV indicates right
ventricle. **P<.05 vs SHR-NF by ANOVA and Tukey's
procedure.
Boluyt et al Gene Expression in the Failing Heart
LV
V 0W
%l #
TGF-BU0LA
185-iw VW
Lane
W
W
1 2 3 4 5 6
LR WKYV
"
#
$
t W
tt WV
7 8 9 10 11 1213
SHR-F-J
SHR-NF----J
RV
TGF-1 .
V0
18isS- 0*
Lane
1 2 3 4 5 6
L-WKY
*
h eto
$0fl
*
7 8 9 10 11 1213 14 15
SHR-NFSHR-F-
12
RV
LV
8
lr
;R.
2
_L
WKY
SHR-NF
SHR-F
WKY
SHR-NF
SHR-F
FIG 8. Levels of transforming growth factor-fl, (TGF-j,8) mRNA
in ventricles of Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) without heart failure (SHR-NF), and SHR with
heart failure (SHR-F). Top, Autoradiogram of Northern blot
analysis. Blots were probed with a cDNA probe for rat TGF-f,1.
Bottom, Bar graphs showing data from Northern blot. Values
represent arbitrary units and are mean+SEM of four to six
samples per group. LV indicates left ventricle; RV, right ventricle.
**P<.05 vs SHR-NF by ANOVA and Tukey's procedure.
Contractile Protein Gene Expression
The decrease in a-MHC mRNA levels in the LV of
failing hearts is in agreement with our previous findings
of a complete disappearance of the V, and V2 myosin
isoforms from LV papillary muscles of failing hearts."
This correspondence between protein and mRNA suggests that the isoform shift is due to transcriptional
regulation or selective destabilization of the a-MHC
mRNA. Although caution must be taken when comparing mRNA values from ventricular wall tissue to protein
measurements made in papillary muscles, the available
evidence suggests that this is reasonable, since papillary
and ventricle wall myosin isoform distributions are
similar in hearts of male Wistar rats,42 and that even
when initial values differ as in aging, the direction and
magnitude of the isoform shift in free wall and papillary
muscle tissue is similar.42 On the basis of the previously
reported shift to 100% V3 myosin isoform in papillary
muscles," we predicted a corresponding increase in
fl-MHC mRNA levels in the LV. There was, however,
no significant increase in f-MHC mRNA levels in either
ventricle of SHR-F. Since the papillary muscles of
WKY rats were comprised of 5:'80% fl-MHC and the
/3-isoform presumably predominated in the ventricles as
29
well, only a small (25% to 40%) increase in f-MHC
mRNA levels would be required to account for the
observed increase in P-MHC protein.1' Thus, it is not
surprising that no significant increase in /3-MHC mRNA
levels was observed.
Since evidence exists that a loss of contractile protein
from the myocardium may contribute to the impaired
function observed in cardiac muscle from failing human
hearts,14 we have determined whether contractile protein mRNA levels are depressed in the SHR model of
heart failure. The present data indicate that levels of
a-cardiac actin, a-skeletal actin, and MLC2 mRNA are
not significantly altered during the transition to failure.
Thus, in the SHR model of heart failure, there is no
grossly detectable downregulation of contractile protein
gene expression. The present study did not directly
address the issue of contractile protein content of failing
myocardium and therefore does not exclude the possibility that there are alterations in protein content that
are due to changes in protein turnover.
Expression of Hypertrophy-Associated Genes
Evidence has accumulated that myocyte dropout due
to myocardial cell death and subsequent hypertrophy of
the remaining viable myocytes occurs in the heart
during aging and hypertension. 15-17 Hypertrophy of
myocytes can be viewed as a physiological adaptation
that serves to normalize wall stress as hemodynamic
load is chronically elevated.9 When hypertrophic adaptive mechanisms fail to adequately normalize wall stress,
dysfunction and overt heart failure ensue. Hallmarks of
hypertrophy include a transient upregulation of ca-skeletal actin gene expression'8 and a sustained elevation in
ANF mRNA levels.22,43 The twofold to threefold higher
levels of a-skeletal actin and ANF mRNA levels observed in SHR-NF compared with WKY rats is consistent with the notion that some myocytes in hearts of
hypertensive rats are exhibiting a nascent hypertrophic
response. It should be noted that the changes in steadystate ANF mRNA levels described here occur in the
context of advanced age, which is also associated with
elevated ANF mRNA levels.44 Despite an additional
elevation in ANF mRNA levels and overt signs of pump
failure, the LVs of SHR-F do not exhibit additional
increases in mass or expression of the a-skeletal actin
gene. In aggregate, these findings suggest that the
capacity for hypertrophy has been reached or exceeded
by some SHR and that an accumulation of connective
tissue, a loss of myocytes, or both limits further adaptation. Factors that may affect the capacity for hypertrophy include absolute cell-size limits imposed by physical characteristics, such as diffusion distance, metabolic
competition for increasingly limited pools of substrate,
and interactions with the extracellular connective tissue
matrix.
SRCA Gene Expression
Studies of human heart tissue suggest a diminished
level of SRCA mRNA in patients with end-stage failure.24 These changes have been postulated to be responsible, in part, for the diastolic dysfunction that characterizes some types of heart failure. It should be noted
that the genetic determinants of hypertension in the
SUR model differ in some respects from factors under-
30
Circulation Research Vol 75, No 1 July 1994
lying essential hypertension in humans,45 raising the
possibility that the mechanisms responsible for heart
failure in humans and SHR may differ in some respects
as well. Expression of the SRCA gene decreases after
aortic constriction in the rat heart and is associated with
depressed sarcoplasmic reticulum pump function.23 In
the SHR model of heart failure, a deficit in the function
of papillary muscles is observed.6,11' 2 In aggregate,
these observations suggest that the expression of the
SRCA gene may be diminished during the transition
from stable hypertrophy to failure. Interestingly, no
decrease was observed in LV SRCA mRNA levels as a
result of either hypertension or during the transition to
failure, although a 24% decrease was observed in the
RV of SHR-NF, with no further decrease during the
transition to failure. Conclusions concerning SRCA
mRNA levels in the LV are limited by the small number
of observations in each group. Thus, although these
results do not exclude the possibility that a small
(<40%) decrease in SRCA mRNA levels during the
transition to failure may occur, they do rule out changes
on the order of magnitude previously observed in
pressure-overload hypertrophy23 and senescence.46,47 A
possible explanation for the lack of a decrease in LV
SRCA mRNA levels during either hypertension or
failure in the present study is that the animals are at an
age when the expression of the SRCA gene is already
depressed in the LV of Wistar rats.46 The aortic constriction-induced downregulation of SRCA gene expression is preserved in hearts of senescent Long-Evans
rats; however, these rats do not exhibit the age-associated decrease in SRCA mRNA levels that occurs in the
Wistar rats.48 The present results in a rat model of heart
failure suggest that SRCA mRNA levels may require
more investigation into the interactive effects of age on
SRCA expression.
Expression of Extracellular
Matrix-Associated Genes
The most striking aspect of the present study is the
threefold to fivefold increase in collagen and fibronectin
mRNA levels between SHR-NF and SHR-F. This suggests that the onset of failure triggers a mechanism that
upregulates fibronectin and collagen gene expression.
Since increases in fibrillar collagen in the interstitium
contribute to tissue stiffness, increases in fibronectin
and collagen gene expression may contribute to impaired function.8 The upregulation of TGF-,f1 gene
expression observed in failing hearts may be consistent
with a stimulatory role for this growth factor, leading to
accumulation of extracellular matrix. Active TGF-f,1
increases fibronectin and collagen expression in a variety of cell types and stimulates their incorporation into
extracellular matrix.25 Villareal and Dillman21 noted a
transient sequential increase in TGF-,31, fibronectin,
and collagen mRNA levels after aortic constriction in
rats, which suggests that activation of TGF-,81 gene
expression is one of the mechanisms by which fibronectin and collagen genes are activated in the heart in vivo.
In contrast to the transient sequential increase in the
expression of extracellular matrix components after
aortic constriction,2' the elevated levels of TGF-f31,
fibronectin, and collagen were evident simultaneously in
hearts of all rats exhibiting signs of failure, suggesting
that increased expression of these genes is sustained in
the failing heart.
Expressions of a number of genes have been investigated for their potential diagnostic value in human
heart failure.24,49 The current results suggest that fibronectin (particularly the EIIIA+ isoform), collagen type
I, collagen type III, and TGF-f31 mRNAs merit similar
evaluation. The EIIIA+ isoform of fibronectin is expressed at high levels during embryogenesis20 and
wound healing19 and in hearts of aortic-constricted,21
SHR,41 and thyroxine-treated50 rats. The structural
properties are not well understood, but it is postulated
that EIIIA+ isoforms of fibronectin may be important
in the deposition of nascent extracellular matrix.'9-21
The current findings are consistent with this notion and
suggest that factors associated with injury and healing
processes may be at work during the transition from
stable hypertrophy to failure.
The finding that mRNAs coding for extracellular
matrix proteins were elevated in both the LV and RV of
failing hearts is consistent with the hypothesis of Weber
and coworkers8,51 that circulating substances may be the
trigger for extracellular matrix production in heart
failure. It should be noted, however, that the magnitude
of RV hypertrophy in SHR-F suggests that load-related
hypertrophy could account for the activation of extracellular matrix production. However, reports that inhibitors of angiotensin-converting enzyme can reverse the
accumulation of fibrotic material52 and prevent the
development of fibrosis and heart failure53 provide
further support for the hypothesis that a circulating
substance may be the trigger. Since cardiac fibroblasts
are the source of collagen types I and III,54 it is
important to identify and characterize factors and conditions that alter production of extracellular matrix
components by these cells. Evidence from studies of
hormonal effects on noncardiac fibroblasts suggests the
possible involvement of angiotensin II, vasopressin, and
aldosterone in modulating gene expression.851 Furthermore, angiotensin II increases proliferation of cultured
cardiac fibroblasts.55,56 Norepinephrine, a physiological
agent known to influence cardiac myocyte hypertrophy
under certain conditions,57 also increases incorporation
of labeled thymidine in cultured cardiac fibroblasts.58
Studies of factors influencing fibronectin and collagen
gene expression in cardiac fibroblasts, as well as potential interactions between myocytes and fibroblasts in the
heart, should provide invaluable insights into the pathophysiology of heart failure.
Acknowledgments
This study was supported by Medical Research Funds from
the Department of Veterans Affairs. Dr Boluyt was supported
by a National Research Council associateship. We thank Ken
Boheler, Kenneth R. Chien, Mon-Li Chu, and Michael Sporn
for generously providing fibronectin, MLC2, collagen, and
TGF-j31 probes, respectively. We are grateful to Kathleen G.
Robinson and Xilin Long for their assistance with technical
aspects of this study and to Paula Wernick for assistance in
preparing the manuscript.
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