© 1997 American Heart Association, Inc.
Articles |
Hemodynamic Regulation of Tumor Necrosis Factor-
Gene and Protein Expression in Adult Feline Myocardium
From the Cardiology and Geriatric Sections of the Department of Medicine, Veterans Administration Medical Center, and Baylor College of Medicine, Houston, Tex.
Correspondence to Douglas L. Mann, MD, Cardiology Research (151C), Room 234, Building 110, VA Medical Center, 2002 Holcombe Blvd, Houston, TX 77030. E-mail dmann{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Abstract Tumor necrosis factor-
(TNF-) mRNA and
protein biosynthesis were examined in adult feline
myocardium in the presence and absence of superimposed
hemodynamic pressure overloading. A brief period of
hemodynamic pressure overloading ex vivo resulted in de
novo TNF- mRNA expression within 30 minutes and de novo TNF-
protein production within 60 minutes; neither TNF- mRNA nor
protein was detected in hearts perfused at normal perfusion pressures.
Moreover, TNF- mRNA and protein biosynthesis were observed in
myocyte and nonmyocyte cell types in the pressure-overloaded
hearts. To determine whether a simple passive stretch of the
myocardium was a sufficient stimulus for TNF-
biosynthesis, we examined TNF- mRNA expression in stretched and
unstretched papillary muscles. This study showed that myocardial
stretch was a sufficient stimulus for the induction of TNF- mRNA
biosynthesis. The functional significance of the intramyocardial
production of TNF- was determined by examining cell motion
in isolated contracting cardiac myocytes treated with
superfusates from pressure-overloaded and control hearts. These
studies showed that cell motion was depressed in myocytes treated with
superfusates from the pressure-overloaded hearts but was normal
with the superfusates from the control hearts. Finally,
hemodynamic pressure overloading in vivo under
physiological conditions was also shown to result
in de novo intramyocardial TNF- mRNA biosynthesis. In conclusion,
this study constitutes the initial demonstration that the adult
mammalian myocardium elaborates biologically active
TNF-, both ex vivo and in vivo, in response to
hemodynamic pressure overloading.
Key Words: tumor necrosis factor- • pressure overload • gene expression • myocyte
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sustained hemodynamic overloading of the heart, whether from a pressure or volume overload, will eventually produce cardiac decompensation in the form of overt congestive failure. Although the elucidation of the mechanisms that are responsible for cardiac decompensation following sustained hemodynamic overloading has remained a central theme of inquiry in cardiovascular research for nearly 50 years, a full description of the maladaptive biochemical mechanisms that are responsible for the transition from compensated cardiac hypertrophy into decompensated congestive failure has not been forthcoming. Important to the present discussion is the recent observation that patients with advanced congestive heart failure and hypertrophic cardiomyopathy express elevated circulating levels of a proinflammatory cytokine named TNF-
.1 2 3 4 5 Although the exact clinical significance of
elevated levels of TNF- in heart failure and hypertrophic
cardiomyopathy remains uncertain, what is quite
clear is that elevated levels of TNF- can produce a number of the
classical features that attend cardiac decompensation, including left
ventricular dysfunction,6 7 8 9 10
cardiomyopathy,11 pulmonary
edema,12 13 14 15 uncoupling of the ß-adrenoceptor from
adenylate cyclase,16 and left
ventricular remodeling.9 10 17
Although the exact source for and site of TNF- production in
heart failure and hypertrophic cardiomyopathy
remain unknown, it is of some interest that recent clinical reports
have shown persistent TNF- mRNA and protein expression in the
ventricles of human subjects with dilated and ischemic
cardiomyopathies.18 19 Interestingly,
explanted hearts from normal organ donors did not express TNF- mRNA
or protein.18 These findings, coupled with the recent
experimental observation that the heart is capable of de novo TNF-
mRNA and protein synthesis after certain forms of
stress,20 21 foreshadowed the interesting possibility that
hemodynamic overloading might serve as a regulatory
mechanism for the induction of TNF- biosynthesis within the
myocardium. Accordingly, in order to address this question,
we systematically examined the effects of hemodynamic
pressure overloading, both ex vivo and in vivo, on TNF- mRNA and
protein biosynthesis in the adult heart. The results of this simple
experimental study constitute the initial demonstration that the adult
mammalian myocardium elaborates biologically active TNF-
in response to hemodynamic pressure overloading.
Moreover, the present study shows that a simple passive stretch of
the myocardium is a sufficient stimulus for TNF- mRNA
biosynthesis in the adult cardiac myocyte. Thus, these studies provide
a potential biophysical link between abnormalities of
hemodynamic overloading and the expression of TNF-
in certain cardiac disease states.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hemodynamic Regulation of TNF-
mRNA Expression
Ex VivoMyocardial TNF-
mRNA production was assessed ex vivo
using a modified Langendorff perfusion
apparatus.6 21 Briefly, adult cats weighing
3.4±0.2 kg, and of random sex, were anesthetized with
ketamine hydrochloride (50 mg/kg IM) and acepromazine
maleate (5 mg/kg IM). After anesthesia, the hearts
were excised rapidly, rinsed thoroughly in chilled (4°C) KHB,
suspended on a cannula, and then perfused at a constant pressure of
80 mm Hg using a roller pump. The initial coronary
superfusate consisted of a nonrecirculating KHB solution of the
following composition (mmol/L): NaCl 130.0, KCl 4.8,
MgSO4 1.2, NaH2PO4 1.2,
CaCl2 0.5, NaHCO3 4.0, glucose 12.5, and HEPES
10.0 (pH 7.4), which was oxygenated with 100%
O2 and maintained at 37°C by a water bath. The heart was
initially perfused for 10 minutes with KHB in a nonrecirculating
fashion to remove any blood products, after which the
apparatus was converted to a recirculating buffer system.
The experimental protocol used for these experiments is illustrated
diagrammatically in Fig 1
. For the
control hearts, the retrograde perfusion pressure was adjusted to a
mean level of 80 mm Hg and maintained at this pressure for the
entire experiment (total perfusion time, 180 minutes). For the
pressure-overloaded hearts, the retrograde perfusion pressure was
initially adjusted to a mean level of 200 mm Hg for 30 minutes,
after which the perfusion pressure was adjusted downward to a mean
pressure of 80 mm Hg for 150 minutes (total perfusion time, 180
minutes). As illustrated in Fig 1, time 0 for the pressure-overloaded
hearts refers to the time immediately after the mean aortic perfusion
pressure was adjusted downward to 80 mm Hg, whereas time 0 for
the control hearts was taken as 30 minutes after perfusion at 80
mm Hg. In an additional control experiment, the retrograde perfusion
pressure was maintained at 200 mm Hg for 180 minutes, in order to
clarify the time course for TNF- mRNA expression after
hemodynamic pressure overloading. A nonworking heart
preparation was used in order to minimize the potential for
ischemia during the superimposition of the pressure
overload.
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To determine the time course of TNF- gene expression in normal or
pressure-overloaded hearts, starting at time 0 and for every 30 minutes
thereafter for a total of 150 minutes, a 1- to 2-g sample of
myocardium was excised from the suspended heart (carefully
sparing the large epicardial vessels). The sample was then frozen
rapidly in liquid nitrogen and stored at -70°C. The positive
controls for these experiments consisted of HL-60 cells stimulated for
180 minutes with bacterial endotoxin (3.2 µmol/L [125
µg/mL]).
Total RNA was extracted from the myocardial samples and the HL-60 cells
by the guanidinium thiocyanate method,22 processed, and
hybridized using a 0.6-kb HindIII-HindIII
fragment of human TNF- (American Tissue Culture Collection) and a
0.5-kb Xba I–HindIII fragment of human GAPDH
exactly as we have described previously.21 The membranes
were washed with 1x SSC and 0.1% SDS at 55°C for 30 minutes,
air-dried, and exposed to Kodak X-Omat A film at -70°C with
intensifying screens.
Hemodynamic Regulation of TNF- Protein
Production Ex Vivo
Myocardial TNF- production was assessed ex vivo using
a modified Langendorff perfusion apparatus, exactly as
described above. After stabilization, the retrograde perfusion pressure
was adjusted to a normal mean pressure of 80 mm Hg in the control
hearts or was increased for 30 minutes to a mean perfusion pressure of
either 150 or 200 mm Hg. At time 0 and for every 30 minutes
thereafter for a total of 150 minutes, a 5-mL sample of the
recirculating KHB was collected to determine TNF- bioactivity, as
assessed by L929 cytotoxicity assay, exactly as we have described
previously.21 Two additional control experiments were
performed to determine whether pressure overloading itself was
responsible for the increase in TNF- protein biosynthesis. First,
immediately before pressure overloading, we performed a ventriculotomy
in the apex of the left ventricle; care was taken not to stretch the
myocardial tissue during the venting of the ventricle. The hearts were
then subjected to 30 minutes of pressure overloading at 200 mm Hg
as described above. Second, we pretreated the isolated hearts for 60
minutes with the antitumoral xanthate compound D609 (0.19
mmol/L), which has been shown to inhibit phospholipase C both in
vitro and in vivo23 24 and to significantly block the
effects of mechanical stretch on c-fos expression in
isolated cardiac myocytes.25 For both of the control
experiments described above, TNF- bioactivity was assessed by L929
cytotoxicity assay at 150 minutes.
Cellular Source for Myocardial TNF- Production In
Vitro
Previous studies from this and other laboratories have shown
that myocytes and nonmyocytes elaborate TNF- in response to
endotoxin provocation.21 26 To determine whether the
cardiac myocyte might also elaborate TNF- after pressure
overloading, we examined the relative production of TNF-
mRNA and protein by myocyte and nonmyocyte cell types isolated
from pressure-overloaded hearts. Hemodynamic pressure
overloading was performed continuously for 180 minutes as described
above. Immediately after pressure overloading, the hearts were
enzymatically digested using techniques standard in this
laboratory.27 28 After enzymatic digestion of the heart,
the cells were rinsed with KHB and gently centrifuged
(200g) in order to separate the myocyte from
nonmyocyte cell types. The resultant cell pellet, which
contains >95% cardiac myocytes, has <2% contaminating
fibroblasts28 29 and <1% contaminating
monocytes.21 The cell pellets and supernatant were then
split into two portions and analyzed for TNF- mRNA and
protein production. Total RNA was extracted and prepared for
Northern analysis as described above, using random-primed DNA
probes for TNF- and GAPDH. Cytosolic TNF- protein was determined
by bioassay exactly as we have described previously.21
Regulation of TNF- Biosynthesis by Passive Stretch of the
Myocardium
To determine whether passive stretch of the
myocardium was a sufficient stimulus for TNF- gene and
protein expression, we isolated papillary muscles from adult cat hearts
and then stretched these linear strips of muscle according to the
general methods described by Peterson and Lesch.30
Briefly, a rapid cardiectomy was performed on adult cats (weighing
2.8±0.1 kg) after anesthesia was induced with
ketamine hydrochloride and acepromazine maleate (as described
above). The infundibulum of the right ventricle was opened and flushed
with KHB solution preequilibrated with 95% O2/5%
CO2 at 37°C, and a papillary muscle of suitable size
(<1-mm2 cross-sectional area) was excised. The
apparatus and procedures used for studying thin strips of
isolated muscle have been described previously in
detail.31 Briefly, the muscles were placed in separate
water-jacketed baths. The base of the papillary muscle was attached to
a Lucite clip that was affixed to the muscle bath; the chordal end of
the muscle was attached to a Lucite clip affixed to a Statham UC-2
force transducer that was positioned directly above the water bath. The
muscle bath solution was kept at 37°C and continuously bubbled with a
mixture of 95% O2/5% CO2 (pH 7.4). Two
platinum plate electrodes were arranged vertically on opposite sides of
the muscle in order to field-stimulate the muscles at a frequency of
0.2 Hz, using a square-wave pulse 5 milliseconds in duration, 20%
above threshold voltage. Outputs were recorded on a polygraph with
an electronic differentiator and collected on a personal computer by a
data-acquisition board. The size of the papillary muscles chosen for
these experiments has been shown to be sufficient for adequate
oxygenation through diffusion at 37°C, with a
stimulation frequency of 0.2 Hz and aeration with 95%
oxygen.32
All muscles were lightly preloaded, such that recognizable contractions were recorded upon field stimulation of each muscle. The thin strips of muscle were allowed to equilibrate for 45 minutes, after which one group of muscles (stretched group) was stretched in a stepwise manner to Lmax. Stress relaxation was allowed to dissipate at each length change. After Lmax was attained, the muscles again were stretched 10% beyond Lmax, such that peak developed tension was clearly decreased from values obtained at Lmax. The stretched group of muscles were maintained at a length that was 10% beyond Lmax for 30 minutes, after which the muscles were returned to lightly preloaded lengths for an additional 90 minutes. Control muscles (unstretched group) were maintained at lightly preloaded lengths for 120 minutes. Both control and stretched papillary muscles were always obtained from the same animal and were studied under identical experimental conditions except for the aforementioned differences in loading conditions. At the end of the experiment, the muscles were carefully excised from the Lucite clips such that only the portions of the myocardium that were free and not clamped between the Lucite clips were used. Muscle specimens were quickly frozen in liquid nitrogen and used for subsequent analysis by RT-PCR as described below.
TNF- mRNA Detection by PCR
TNF- mRNA in the stretched and unstretched papillary muscles
was detected using PCR amplification, according to the methods
described by Ma et al.33 The
oligonucleotide primers were chosen on the basis of
unique DNA sequences published for feline TNF- (Genbank sequence,
FDTNFA). The sense primer (5'-CTTCTCGAACTCCGAGTGACAAGCC-3') was
derived from bases 239 to 254 of the coding region of TNF-; the
antisense primer used (5'-TGATGGCGTGGGTGAGGAGCACATG-3') was derived
from bases 445 to 470 of the coding region of TNF-. PCR
amplification of total mRNA from the heart was performed using
conditions and parameters identical to those that we have
described previously21 and was carried out for 30 cycles.
The PCR reaction products were separated on a 1.5% agarose gel,
stained with ethidium bromide, and visualized by UV light.
Myocardial TNF- mRNA and Protein Biosynthesis In Vivo
In order to confirm the physiological
relevance of the findings obtained in the ex vivo nonworking feline
heart model, we next asked whether the imposition of a
physiologically relevant
hemodynamic pressure overload in vivo would stimulate
TNF- mRNA and protein biosynthesis. Briefly, adult cats of random
sex, weighing 3.1±0.2 kg, were subjected to left
ventricular pressure overloading by partially occluding the
ascending aorta with an elastic band. Briefly, cats were
anesthetized with ketamine hydrochloride (15
mg/kg IM), meperidine (2.2 mg/kg IM), and succinylcholine
(1 mg/kg IV) and placed on a positive-pressure respirator. A
median sternotomy was performed, and an elastic ligature was placed
around the ascending aorta, above the level of the coronary
arteries but below the innominate artery. Left ventricular
pressure overloading was achieved by tightening the ligature around the
aorta for 30 minutes, in order to achieve a mean gradient of 
45
mm Hg; for the sham-operated cats, the elastic ligature was left in
place around the aorta but was not tightened. Throughout these
experiments, care was taken to leave the pericardium intact in both
pressure-overloaded and sham-operated cats. After 30 minutes, the
ligature was removed from the pressure-overloaded and the sham-operated
cats, and the animals were allowed to recover for an additional 90
minutes before euthanasia under deep anesthesia. At this
time (total time, 120 minutes), the heart was removed and washed three
times in chilled PBS (4°C), and the left ventricular free
wall was frozen in liquid nitrogen. Total RNA was prepared as described
above.
Left ventricular pressures were obtained with a fluid-filled catheter attached to a strain gauge, which was coupled to a 22-gauge needle that was inserted through the apex of the left ventricle. Systemic pressures were monitored by a fluid-filled catheter attached to a strain gauge that was positioned in the proximal left common carotid artery. The midchest position was taken as a zero reference point for pressure measurements.
Biological Activity of Myocardial TNF-
TNF-–Induced Cytotoxicity
The methods for assessing TNF-–induced cytotoxicity by L929
bioassay have been described previously in
detail.21 34
TNF-–Induced Negative Inotropic Effects
The presence or absence of TNF- in the superfusates
of the normal-pressure and pressure-overloaded hearts was also assessed
using a simple cell motion assay, exactly as described
previously.6 21 34 Briefly, adult feline cardiac myocytes
were freshly isolated,27 28 29 allowed to stabilize for 1
hour, and treated for 30 minutes at 37°C with a 1:2 dilution of
superfusate from either control or pressure-overloaded hearts.
The appropriate controls for these studies consisted of myocytes
treated with diluent alone (0.1% human serum albumin) or 100
U/mL rhTNF- (Genzyme Corp). To confirm the specificity of any
superfusate-induced effects on cell motion, the 1:2 dilution of
the superfusate from the pressure-overloaded heart was
preincubated for 60 minutes with 1 mL/L (1 µL/mL) of a
neutralizing polyclonal rabbit anti-human TNF-, as we have described
previously.6 Cell motion was characterized by video-edge
detection, at a stimulation frequency of 0.25 Hz, using experimental
conditions identical to those that we have described
elsewhere.6 34 For these studies, we examined the percent
change in cell length. In order to compare results between different
myocyte isolations, the values for cell shortening were expressed as a
"fold change" in the amplitude of cell shortening compared with
control values from that same myocyte isolation. Thus, for each of the
studies described above, there was always an appropriate control group
that was obtained from the same primary myocyte isolation.
Statistics
Value are expressed as mean±SE. A nonpaired t
test was used to examine TNF- levels in diluent and pressure-treated
hearts ex vivo and in vivo and to compare
hemodynamic data in pressure-overloaded and
sham-operated animals. One-way ANOVA was used to test for mean
differences in cell shortening after treatment with rhTNF- or with
superfusates from the control or pressure-overloaded hearts;
where appropriate, post hoc multiple comparison testing (Dunnett's
test) was performed to test for differences between control and
experimental groups. Significant differences were said to exist at a
value of P<.05.
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Results |
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Hemodynamic Regulation of TNF-
mRNA
Production Ex VivoFig 2
illustrates two important
findings with respect to the hemodynamic regulation of
TNF- mRNA biosynthesis. First, as shown in Fig 2A, TNF- mRNA was
not constitutively expressed in a freshly excised, buffer-perfused
heart that was maintained at a normal perfusion pressure of 80
mm Hg for 180 minutes. Similar qualitative findings with respect to
TNF- mRNA expression were observed in two additional hearts perfused
at 80 mm Hg. In contrast to the findings obtained in hearts
perfused at 80 mm Hg, Fig 2B shows that TNF- mRNA expression
was evident within 30 minutes after the onset of
hemodynamic pressure overloading. As illustrated,
TNF- gene expression peaked between 30 and 60 minutes after
hemodynamic pressure overloading and then decreased
rapidly toward baseline within 150 minutes. Similar qualitative
findings with respect to TNF- mRNA expression were observed in two
additional hearts perfused at 200 mm Hg for 30 minutes. In order
to determine whether the observed decrease in TNF- mRNA expression
was the result of recovery from hemodynamic pressure
overloading, we examined TNF- mRNA expression in hearts subjected to
180 minutes of continuous pressure overloading. As shown in Fig 2B, TNF- mRNA levels remained elevated when the
hemodynamic pressure overloading was sustained. Thus,
when continuous pressure overloading was applied, there was robust
TNF- mRNA expression.
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Hemodynamic Regulation of TNF- Protein
Production Ex Vivo
The inset of Fig 3 illustrates the
time course for the appearance of TNF- bioactivity in a
representative buffer-perfused heart subjected to a
brief 30-minute period of hemodynamic pressure
overloading (200 mm Hg). As shown, TNF- bioactivity was not
detectable in the myocardial superfusate immediately after the
cessation of hemodynamic pressure overloading (time 0)
but was detectable as early as 60 minutes after
hemodynamic overloading and peaked at 90 to 120
minutes. TNF- bioactivity in the superfusates from the
pressure-overloaded heart could be completely neutralized at each of
the time points tested by pretreating the superfusate with a
polyclonal antibody directed against TNF-.21 Fig 3
depicts the results of group data, obtained at 150 minutes, for hearts
perfused at pressures of 80 mm Hg (n=5), 150 mm Hg (n=6),
and 200 mm Hg (n=8). The important finding shown is that whereas
TNF- bioactivity was not detected in the control hearts perfused at
80 mm Hg, TNF- bioactivity was detected in the
superfusates from the hearts subjected to a 150 mm Hg and
200 mm Hg pressure overload. The level of TNF- bioactivity
observed after perfusion at 200 mm Hg was significantly different
(P<.05) from control values, whereas the level of TNF-
after perfusion at 150 mm Hg was not significant statistically.
To determine whether pressure overloading was responsible for the
increase in TNF- protein biosynthesis, we performed an apical left
ventriculotomy immediately before the onset of
hemodynamic pressure overloading. As shown in Fig 3, venting the left ventricle (n=4) to atmospheric pressure before the
onset of pressure overloading completely abrogated the effect of
pressure overloading on TNF- protein biosynthesis (P>.05
compared with control), suggesting that intracavitary pressure
overloading was important for TNF- protein biosynthesis. Finally, as
an additional control experiment, we also pretreated the intact hearts
with 0.19 mmol/L D609 (n=4), an inhibitor of
the phospholipase C pathway,23 24 since this pathway has
been shown to be important in mediating the effects of mechanical
stretch on c-fos expression in isolated cardiac
myocytes.25 Fig 3 shows that inhibiting the phospholipase
C pathway blocked the effects of hemodynamic pressure
overloading on TNF- protein biosynthesis (P>.05 compared
with control), suggesting that the effects of
hemodynamic overloading were mediated through a
stretch-activated signaling pathway.
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) for a representative heart
perfused at 200 mm Hg; TNF-
). The bar graph depicts the values obtained for group data for
hearts perfused at 80 mm Hg (open bar, n=5 hearts), 150
mm Hg (stippled bar, n=6 hearts), and 200 mm Hg (solid bar, n=8
hearts) and for hearts that were vented (left hatched bar, n=4 hearts)
and hearts that were pretreated with D609 (right hatched bar, n=4
hearts) before perfusion at 200 mm Hg. *P<.05
compared with hearts perfused at 80 mm Hg.
Cellular Source for Myocardial TNF- Production Ex
Vivo
Fig 4 compares and contrasts the
relative production of TNF- mRNA (n=3) and protein by
cardiac myocytes (n=8 dishes) and nonmyocyte cell types 9 (n=8
dishes) in the heart after hemodynamic pressure
overloading. As shown in Fig 4 on the right, the supernatant from the
cell isolation, which is predominately composed of nonmyocyte
cell types (>95%), expressed both TNF- mRNA and protein. In Fig 4
on the left, the cell pellets, which are predominantly composed
(>95%) of cardiac myocytes, expressed both TNF- mRNA and protein,
although the relative amount of TNF- mRNA in the cell pellet
appeared less compared with that observed in the cell supernatant.
Thus, both nonmyocyte and myocyte cell types express TNF-
mRNA and protein after hemodynamic pressure
overloading.
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Regulation of TNF- mRNA and Protein Biosynthesis by Passive
Stretch
To determine whether passive stretch of the myocardium
was a sufficient stimulus for the induction of TNF- biosynthesis, we
examined TNF- gene expression in stretched and unstretched papillary
muscles. Fig 5 shows that whereas TNF-
mRNA was not detectable by RT-PCR in an unstretched papillary muscle,
TNF- mRNA was detectable by RT-PCR in a papillary muscle that had
been stretched 10% beyond Lmax for 30 minutes; the far
right lane depicts the RT-PCR product obtained from human TNF-
DNA and serves as a positive control. As shown, TNF- mRNA PCR
products were not detectable in the stretched or unstretched
papillary muscles in the absence of RT, thereby excluding the presence
of DNA contamination. Similar results were obtained in three additional
unstretched and three additional stretched papillary muscles from two
different animals.
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Functional Significance of Cardiac TNF- Production
To determine whether the amount of TNF- that was produced in
response to the superimposition of a hemodynamic
pressure overload was sufficient to affect cardiac myocyte cell motion,
we incubated freshly isolated cardiac myocytes for 30 minutes with
superfusates from the normal and pressure-overloaded hearts. As
shown in Fig 6, when freshly
isolated cardiac myocytes were incubated with a 1:2 dilution of the
superfusate from the hearts perfused at normal pressures, there
was no effect on cell motion. However, when the cells were incubated
with a 1:2 dilution of the superfusate from the
pressure-overloaded heart (200 mm Hg), there was an 30%
decrease in the extent of cell shortening. As shown, the depression in
cell shortening was similar to that observed after stimulation with
recombinant human TNF- (100 U/mL). Moreover, the negative inotropic
effects of the superfusate from the pressure-overloaded hearts
could be completely abrogated by pretreatment with a neutralizing
polyclonal TNF- antibody. Since we incubated the myocytes with
superfusates from the hearts for only 30 minutes, we cannot
exclude the possibility that other cytokines/peptides, whose
time course of action was longer, may also have been present. ANOVA
indicated that the overall differences in cell shortening among groups
were significantly different (P<.001); post hoc ANOVA
testing (Dunnett's test) indicated that the decrease in the extent of
cell shortening for cells treated with the 1:2 dilution of the
superfusate from the pressure-overloaded hearts or recombinant
human TNF- was significantly different from control
(P<.05), whereas the extent of cell shortening for the
cells treated with superfusate from the hearts perfused at
normal pressures or the superfusate retreated with a
neutralizing polyclonal antibody was not significantly different from
control (P>.05).
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Myocardial TNF- Biosynthesis In Vivo
The Table summarizes three important
characteristics of the animals that were used in the in vivo studies
designed to examine hemodynamic regulation of TNF-
gene and protein biosynthesis. First, there was no significant
difference in either the systolic (P=.86) or
diastolic (P=.84) blood pressure at baseline in
the sham-operated and pressure-overloaded animals. Second, aortic
banding resulted in a significant 46±5.5 mm Hg mean pressure
gradient across the left ventricle, whereas there was no change in the
gradient across the left ventricle in the sham-operated animals. Third,
immediately after the aortic band was removed, there was no significant
difference in either the systolic (P=.21) or
diastolic (P=.25) left ventricular
pressures compared with values that were obtained at baseline, thus
suggesting that an abrupt pressure overload did not produce
functionally significant ischemic injury to the left ventricle
in the banded cats.
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To determine whether pressure overloading would provoke myocardial
TNF- gene expression in vivo, we examined cardiac TNF- mRNA
biosynthesis. In Fig 7, panels A and B
depict a representative hemodynamic
left ventricular pressure tracing from a sham-operated cat
(A) and RT-PCR products for TNF- mRNA from three consecutive
sham-operated cats (B). As shown, under normal
hemodynamic loading conditions in vivo, TNF- mRNA
(Fig 7B) was not detectable in the myocardium by PCR (30
cycles). In contrast to the findings obtained with normal
hemodynamic loading conditions, panels C and D show
that when an abrupt hemodynamic pressure overload
(45 mm Hg) was superimposed on the left ventricle (C), TNF-
mRNA was expressed de novo within 120 minutes (D) in myocardial samples
obtained from three consecutive banded animals. Thus,
hemodynamic pressure overloading in vivo is sufficient
to provoke TNF- mRNA biosynthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This simple experimental study, in which the effects of hemodynamic overloading were systematically studied ex vivo and in vivo, shows that the adult mammalian heart efficiently synthesizes biologically active TNF-
after
hemodynamic pressure overloading. Five distinct, but
mutually complementary, lines of evidence support this statement.
First, when freshly isolated buffer-perfused hearts were subjected to a
brief (30-minute) period of pressure overloading, TNF- mRNA was
expressed de novo within 30 minutes (Fig 2B
), whereas TNF- mRNA was
not detectable at any time point in the hearts perfused at normal
perfusion pressures (Fig 2A). Second, there was a striking increase in
the elaboration of biologically active TNF- in the
superfusates from the pressure-overloaded hearts (Fig 3),
whereas TNF- bioactivity was not evident in the superfusates
from the diluent stimulated hearts (Fig 3). As shown, TNF-
bioactivity was evident as early as 60 minutes and peaked 90 to 120
minutes after cessation of hemodynamic pressure
overloading (Fig 3, insert). The specificity of the effects of
hemodynamic overloading on TNF- protein biosynthesis
was demonstrated by the observation that venting the left ventricle
before the imposition of pressure overloading completely abrogated the
load-induced increase in TNF- protein biosynthesis (Fig 3). Third,
TNF- mRNA and protein were found to be produced by both myocyte and
nonmyocyte cell types residing within the heart (Fig 4), as we
have reported previously in studies of feline myocardium
after endotoxin stimulation.21 Fourth, when isolated
cardiac myocytes were exposed to the superfusates (1:2
dilution) from pressure-overloaded hearts, cell motion was depressed to
a degree similar to that observed when adult cardiac myocytes were
treated with recombinant human TNF- (Fig 6), whereas
superfusates from hearts perfused at normal pressures had no
effect on cell motion. Moreover, the negative inotropic effect of the
superfusates from the pressure-overloaded hearts could be
abrogated completely by pretreating the superfusates with a
neutralizing anti-TNF- antibody. Finally, to confirm the
physiological relevance of the ex vivo studies
presented herein, we also examined the effects of an abrupt
hemodynamic pressure overload in vivo. These latter
studies confirmed our ex vivo findings, in that we again observed de
novo expression of intramyocardial TNF- mRNA (Fig 7D) in vivo in the
pressure-overloaded animals, whereas intramyocardial TNF- mRNA (Fig 7B) was not observed in the sham-operated animals. Importantly, the
degree of hemodynamic overloading obtained in vivo
occurred with an intact pericardium and did not produce appreciable
ischemic damage to the left ventricle, as suggested by the
complete normalization of left ventricular systolic
and diastolic pressures immediately following the removal
of the left ventricular pressure overload as well as by the
absence of ventricular arrhythmias in the banded
animals. Moreover, the coronary arterial tree was
included in the hypertensive circuit in our model of
supravalvular aortic banding, which would tend to minimize the
development of subendocardial ischemia during
hemodynamic pressure overloading. Taken together, these
observations constitute the initial demonstration that the adult
mammalian myocardium synthesizes biologically active
TNF- after hemodynamic pressure overloading.
A second important finding of the present study was the observation
that a simple passive stretch of the myocardium was a
sufficient stimulus for TNF- mRNA biosynthesis in adult mammalian
myocardium (Fig 5). On the basis of the above findings with
respect to TNF- biosynthesis ex vivo and in vivo, we considered that
"stretching" of the myocardium might be an important
biophysical stimulus for TNF- mRNA and protein biosynthesis. To test
this hypothesis, we examined the presence or absence of TNF- mRNA
and protein biosynthesis in isolated thin strips of myocardial tissue
that were either allowed to remain at their preloaded length or were
stretched 10% beyond Lmax. This simple study showed that a
brief passive stretch of the myocardium was a sufficient
stimulus for de novo TNF- mRNA expression. A second important line
of evidence that implicates mechanical stretch as an important
mechanism for TNF- biosynthesis was shown by the studies in which
inhibiting the phospholipase C pathway with D60923 blocked
the effects of pressure overloading on TNF- protein biosynthesis.
The rationale for inhibiting this pathway was based on a previous study
in which inhibition of the phospholipase C pathway with D609 blocked
the effects of mechanical stretch on c-fos expression in
neonatal cardiac myocytes.25 Fig 3 shows that when the
phospholipase C pathway was blocked before the imposition of pressure
overload, TNF- protein biosynthesis was abrogated completely (Fig 3). Taken together, these studies provide a potential biophysical link
between abnormalities of hemodynamic overloading and
the expression of TNF- in certain cardiac disease states.
Regulation of TNF- mRNA and Protein Biosynthesis in the
Adult Heart
Although elevated levels of TNF- were first reported in
patients with advanced congestive heart failure,1
subsequent studies have identified elevated levels of this
proinflammatory cytokine in a variety of cardiac disease states
including acute viral myocarditis,35 36 cardiac allograft
rejection,37 38 myocardial infarction,39 40
myocardial reperfusion injury,41 and hypertrophic
cardiomyopathy.4 Although it is
generally thought that activation of the immune system is responsible
for TNF- production in cardiovascular
disorders that are canonically associated with myocardial inflammation,
such as acute viral myocarditis and cardiac allograft rejection, the
stimulus for and source of TNF- production in conditions
that are not traditionally associated with immune activation, such as
heart failure and hypertrophic cardiomyopathy,
remain relatively less certain. Germane to this discussion is the
observation that the adult heart is capable of efficient TNF- mRNA
and protein biosynthesis in response to certain forms of
stress.20 21 Although the literature with respect to
TNF- gene regulation in the adult heart is limited at present,
at least three important themes have emerged thus far. First, neither
TNF- mRNA nor TNF- protein appears to be constitutively expressed
in the unstressed adult mammalian heart.20 21 Second, both
TNF- mRNA and protein are rapidly synthesized by the heart in
response to an appropriate stressful stimulus.20 21 Third,
once TNF- mRNA biosynthesis is initiated, myocardial TNF- mRNA
levels return rapidly toward baseline after removal of the inciting
stress.21 Taken together, the above experimental studies
suggest that in the normal adult heart, TNF- gene and protein
expression are self-limited and occur only in relation to a
superimposed environmental stress. It is therefore of some interest
that in a recent analysis of clinical material, we have shown
that TNF- mRNA and protein were both expressed in failing human
hearts, whereas neither TNF- mRNA nor protein was detected in
nonfailing human hearts.18 Thus, for reasons that are
unclear at present, both TNF- mRNA and protein appear to be
persistently expressed in the failing heart. Although direct
correlations between the experimental effects observed in thin strips
of myocardial tissue and the complex effects of TNF- gene regulation
in the failing heart are not appropriate, the results of the
present study do suggest the intriguing possibility that a passive
myocardial stretch, such as occurs during the process of progressive
left ventricular remodeling, may be a sufficient stimulus
to provoke persistent TNF- gene expression in the failing human
heart.
Conclusion
The present study constitutes the initial demonstration that
hemodynamic pressure overloading is a sufficient
stimulus for TNF- mRNA and protein biosynthesis in the adult
mammalian heart. An important implication of these findings is that
load-induced TNF- biosynthesis may represent a novel
autocrine and/or paracrine mechanism for regulating cardiac structure
and function in health and disease. Support for this statement is
implicit in a recent study from this laboratory showing that
concentrations of TNF- observed after hemodynamic
pressure overloading are sufficient to increase protein synthesis in
adult cardiac myocytes42 as well as a recent study in
which stimulation of cultured neonatal rat cardiac myocytes with
interleukin-1ß was shown to provoke the "heart failure
phenotype," including downregulation of the calcium-ATPase
gene as well as upregulation of the gene for atrial
natriuretic factor.43 Given that the
expression of interleukin-1ß is regulated directly by
TNF-,44 one intriguing (albeit speculative) possibility
is that prolonged and/or excessive hemodynamic
overloading may produce maladaptive changes in the heart by activating
a hierarchical cascade of proinflammatory cytokines.
Although the above statements have focused attention on the potential
deleterious effects of TNF-, the coordinated and tightly regulated
expression of TNF- mRNA and protein synthesis in the heart has
prompted us to suggest that short-term expression of myocardial TNF-
may confer some as-yet-unknown survival benefit to the
host.21 45 Indeed, given that TNF-like activity has been
identified in simple protostome invertebrate phyla whose origins date
back to the Cambrian period,46 47 it would probably be
naive to suggest that nature has conserved TNF- for nearly 600
million years entirely for the purpose of provoking maladaptive changes
in multicellular organisms. Although the potential salutary effects of
TNF- in the heart remain largely unknown at present, the
expression of low concentrations of TNF- for relatively brief
periods of time may provide the heart with a short-term adaptive
response to environmental stress, both by increasing sarcomeric protein
synthesis in the adult cardiac myocyte42 and by increasing
the expression of heat shock proteins in these cells.21 48
However, with sustained environmental stress, such as might be expected
to occur after prolonged hemodynamic overloading, we
postulate that the short-term beneficial effects of TNF- are
contravened and ultimately outweighed by the known untoward effects of
sustained proinflammatory cytokine expression, including left
ventricular dysfunction,6 7 8 9 10 pulmonary
edema,12 13 14 15 left ventricular
remodeling,9 10 17 uncoupling of the ß-adrenoceptor from
adenylate cyclase,16 generation of free
radicals,49 50 and cardiac myocyte
apoptosis,51 52 53 any or all of which may
directly contribute to the overt manifestations of cardiac
decompensation.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This study was supported by research funds from the Department of Veterans Affairs and the National Institutes of Health (P50 HL-O6H). The authors gratefully acknowledge the technical assistance of Dorellyn Lee-Jackson.
Received January 21, 1997; accepted May 27, 1997.
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References |
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 V. Todorov, M. Muller, F. Schweda, and A. Kurtz Tumor necrosis factor-alpha inhibits renin gene expression Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2002; 283(5): R1046 - R1051. [Abstract] [Full Text] [PDF]
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 M. Afanasyeva and N. R. Rose Cardiomyopathy Is Linked to Complement Activation Am. J. Pathol., August 1, 2002; 161(2): 351 - 357. [Full Text] [PDF]
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J. G.F Bronzwaer, C. Zeitz, C. A Visser, and W. J Paulus Endomyocardial nitric oxide synthase and the hemodynamic phenotypes of human dilated cardiomyopathy and of athlete's heart Cardiovasc Res, August 1, 2002; 55(2): 270 - 278. [Abstract] [Full Text] [PDF]
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G. Wright, I. S. Singh, J. D. Hasday, I. K. Farrance, G. Hall, A. S. Cross, and T. B. Rogers Endotoxin stress-response in cardiomyocytes: NF-kappa B activation and tumor necrosis factor-alpha expression Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H872 - H879. [Abstract] [Full Text] [PDF]
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W. S. Bradham, B. Bozkurt, H. Gunasinghe, D. Mann, and F. G. Spinale Tumor necrosis factor-alpha and myocardial remodeling in progression of heart failure: a current perspective Cardiovasc Res, March 1, 2002; 53(4): 822 - 830. [Abstract] [Full Text] [PDF]
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 D.L. MANN The Yin/Yang of Innate Stress Responses in the Heart Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 363 - 370. [Abstract] [PDF]
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P. O. Iversen, P. R. Woldbaek, T. Tonnessen, and G. Christensen Decreased hematopoiesis in bone marrow of mice with congestive heart failure Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R166 - R172. [Abstract] [Full Text] [PDF]
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P. O. Iversen, G. Nicolaysen, and M. Sioud DNA enzyme targeting TNF-alpha mRNA improves hemodynamic performance in rats with postinfarction heart failure Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2211 - H2217. [Abstract] [Full Text] [PDF]
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S. Adamopoulos, J. T. Parissis, and D. Th. Kremastinos A glossary of circulating cytokines in chronic heart failure Eur J Heart Fail, October 1, 2001; 3(5): 517 - 526. [Abstract] [Full Text] [PDF]
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C. Stamm, I. Friehs, D. B. Cowan, A. M. Moran, H. Cao-Danh, L. F. Duebener, P. J. del Nido, and F. X. McGowan Jr Inhibition of Tumor Necrosis Factor-{alpha} Improves Postischemic Recovery of Hypertrophied Hearts Circulation, September 18, 2001; 104 (2009): I-350 - I-355. [Abstract] [Full Text] [PDF]
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N. Sivasubramanian, M. L. Coker, K. M. Kurrelmeyer, W. R. MacLellan, F. J. DeMayo, F. G. Spinale, and D. L. Mann Left Ventricular Remodeling in Transgenic Mice With Cardiac Restricted Overexpression of Tumor Necrosis Factor Circulation, August 14, 2001; 104(7): 826 - 831. [Abstract] [Full Text] [PDF]
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T. Kadokami, C. F. McTiernan, T. Kubota, C. S. Frye, G. S. Bounoutas, P. D. Robbins, S. C. Watkins, and A. M. Feldman Effects of soluble TNF receptor treatment on lipopolysaccharide-induced myocardial cytokine expression Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2281 - H2291. [Abstract] [Full Text] [PDF]
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S. F. Nagueh, S. J. Stetson, N. M. Lakkis, D. Killip, A. Perez-Verdia, M. L. Entman, W. H. Spencer III, and G. Torre-Amione Decreased Expression of Tumor Necrosis Factor-{{alpha}} and Regression of Hypertrophy After Nonsurgical Septal Reduction Therapy for Patients With Hypertrophic Obstructive Cardiomyopathy Circulation, April 10, 2001; 103(14): 1844 - 1850. [Abstract] [Full Text] [PDF]
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F. Wang, Y. Seta, G. Baumgarten, D. J. Engel, N. Sivasubramanian, and D. L. Mann Functional Significance of Hemodynamic Overload-Induced Expression of Leukemia-Inhibitory Factor in the Adult Mammalian Heart Circulation, March 6, 2001; 103(9): 1296 - 1302. [Abstract] [Full Text] [PDF]
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S. J. Zieman, G. Gerstenblith, E. G. Lakatta, G. O. Rosas, K. Vandegaer, K. M. Ricker, and J. M. Hare Upregulation of the Nitric Oxide-cGMP Pathway in Aged Myocardium : Physiological Response to l-Arginine Circ. Res., January 19, 2001; 88(1): 97 - 102. [Abstract] [Full Text] [PDF]
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 B. Bozkurt Activation of cytokines as a mechanism of disease progression in heart failure Ann Rheum Dis, November 1, 2000; 59(90001): i90 - 93. [Full Text] [PDF]
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D. Kalra, G. Baumgarten, Z. Dibbs, Y. Seta, N. Sivasubramanian, and D. L. Mann Nitric Oxide Provokes Tumor Necrosis Factor-{alpha} Expression in Adult Feline Myocardium Through a cGMP-Dependent Pathway Circulation, September 12, 2000; 102(11): 1302 - 1307. [Abstract] [Full Text] [PDF]
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E. J. Birks, V. J. Owen, P. B. J. Burton, A. E. Bishop, N. R. Banner, A. Khaghani, J. M. Polak, and M. H. Yacoub Tumor Necrosis Factor-{alpha} Is Expressed in Donor Heart and Predicts Right Ventricular Failure After Human Heart Transplantation Circulation, July 18, 2000; 102(3): 326 - 331. [Abstract] [Full Text] [PDF]
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S. R. Kapadia, K. Yakoob, S. Nader, J. D. Thomas, D. L. Mann, and B. P. Griffin Elevated circulating levels of serum tumor necrosis factor-alpha in patients with hemodynamically significant pressure and volume overload J. Am. Coll. Cardiol., July 1, 2000; 36(1): 208 - 212. [Abstract] [Full Text] [PDF]
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 P.P. Davey and H. Ashrafian New therapies for heart failure: is thalidomide the answer? QJM, May 1, 2000; 93(5): 305 - 311. [Abstract] [Full Text] [PDF]
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M. N. Sack, R. M. Smith, and L. H. Opie Tumor necrosis factor in myocardial hypertrophy and ischaemia -- an anti-apoptotic perspective Cardiovasc Res, February 1, 2000; 45(3): 688 - 695. [Full Text] [PDF]
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W. J. Paulus How are cytokines activated in heart failure? Eur J Heart Fail, December 17, 1999; 1(4): 309 - 312. [Full Text] [PDF]
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G. Torre-Amione, S. J. Stetson, K. A. Youker, J.-B. Durand, B. Radovancevic, R. M. Delgado, O. H. Frazier, M. L. Entman, and G. P. Noon Decreased Expression of Tumor Necrosis Factor-{alpha} in Failing Human Myocardium After Mechanical Circulatory Support : A Potential Mechanism for Cardiac Recovery Circulation, September 14, 1999; 100(11): 1189 - 1193. [Abstract] [Full Text] [PDF]
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D. L. Mann Mechanisms and Models in Heart Failure : A Combinatorial Approach Circulation, August 31, 1999; 100(9): 999 - 1008. [Full Text] [PDF]
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M. R. Bergman, R. H. Kao, S. A. McCune, and B. J. Holycross Myocardial tumor necrosis factor-alpha secretion in hypertensive and heart failure-prone rats Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H543 - H550. [Abstract] [Full Text] [PDF]
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T. Yokoyama, K. Sekiguchi, T. Tanaka, K. Tomaru, M. Arai, T. Suzuki, and R. Nagai Angiotensin II and mechanical stretch induce production of tumor necrosis factor in cardiac fibroblasts Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H1968 - H1976. [Abstract] [Full Text] [PDF]
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D. R. Wagner, T. Kubota, V. J. Sanders, C. F. McTiernan, and A. M. Feldman Differential regulation of cardiac expression of IL-6 and TNF-alpha by A2- and A3-adenosine receptors Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H2141 - H2147. [Abstract] [Full Text] [PDF]
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S. Sasayama, A. Matsumori, and Y. Kihara New insights into the pathophysiological role for cytokines in heart failure Cardiovasc Res, June 1, 1999; 42(3): 557 - 564. [Full Text] [PDF]
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 E. H. Herrera-Garza, S. J. Stetson, A. Cubillos-Garzon, M. T. Vooletich, J. A. Farmer, and G. Torre-Amione Tumor Necrosis Factor-{alpha}: A Mediator of Disease Progression in the Failing Human Heart Chest, April 1, 1999; 115(4): 1170 - 1174. [Abstract] [Full Text] [PDF]
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M. W. Irwin, S. Mak, D. L. Mann, R. Qu, J. M. Penninger, A. Yan, F. Dawood, W.-H. Wen, Z. Shou, and P. Liu Tissue Expression and Immunolocalization of Tumor Necrosis Factor-{alpha} in Postinfarction Dysfunctional Myocardium Circulation, March 23, 1999; 99(11): 1492 - 1498. [Abstract] [Full Text] [PDF]
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A. Nicoletti and J.-B. Michel Cardiac fibrosis and inflammation: interaction with hemodynamic and hormonal factors Cardiovasc Res, March 1, 1999; 41(3): 532 - 543. [Abstract] [Full Text] [PDF]
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D. Bryant, L. Becker, J. Richardson, J. Shelton, F. Franco, R. Peshock, M. Thompson, and B. Giroir Cardiac Failure in Transgenic Mice With Myocardial Expression of Tumor Necrosis Factor-{alpha} Circulation, April 14, 1998; 97(14): 1375 - 1381. [Abstract] [Full Text] [PDF]
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D. R. Wagner, C. McTiernan, V. J. Sanders, and A. M. Feldman Adenosine Inhibits Lipopolysaccharide-Induced Secretion of Tumor Necrosis Factor-{alpha} in the Failing Human Heart Circulation, February 17, 1998; 97(6): 521 - 524. [Abstract] [Full Text] [PDF]
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E. A. Palmieri, G. Benincasa, F. Di Rella, C. Casaburi, M. G. Monti, G. De Simone, L. Chiariotti, L. Palombini, C. B. Bruni, L. Sacca, et al. Differential expression of TNF-alpha , IL-6, and IGF-1 by graded mechanical stress in normal rat myocardium Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H926 - H934. [Abstract] [Full Text] [PDF]
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D. Kalra, N. Sivasubramanian, and D. L. Mann Angiotensin II Induces Tumor Necrosis Factor Biosynthesis in the Adult Mammalian Heart Through a Protein Kinase C-Dependent Pathway Circulation, May 7, 2002; 105(18): 2198 - 2205. [Abstract] [Full Text] [PDF]
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G. Baumgarten, P. Knuefermann, D. Kalra, F. Gao, G. E. Taffet, L. Michael, P. J. Blackshear, E. Carballo, N. Sivasubramanian, and D. L. Mann Load-Dependent and -Independent Regulation of Proinflammatory Cytokine and Cytokine Receptor Gene Expression in the Adult Mammalian Heart Circulation, May 7, 2002; 105(18): 2192 - 2197. [Abstract] [Full Text] [PDF]
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