Published online before print July 5, 2001, doi: 10.1161/hh1401.093293
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© 2001 American Heart Association, Inc.
Article |
UT-A Urea Transporter Protein in Heart
Increased Abundance During Uremia, Hypertension, and Heart Failure
Presented in part at Experimental Biology ’99, Washington, DC, April 17–21, 1999, the 33rd Annual Meeting of the American Society of Nephrology, Toronto, Ontario, Canada, October 13–16, 2000, and Experimental Biology 2001, Orlando, Fla, March 31–April 4, 2001, and published in abstract form (FASEB J. 1999;13:A392; J Am Soc Nephrol. 2000;11:24A; and FASEB J. 2001;15:A852).
From the Renal Division, Department of Medicine (R.D., J.D.K., J.J.D., P.R., B.R.R., J.M.S.) and Department of Physiology (J.M.S.), Emory University School of Medicine, Atlanta, Ga, and Department of Physiology and Biophysics (J.B.V., A.A.M.), USC Keck School of Medicine, Los Angeles, Calif.
Correspondence to Dr Jeff M. Sands, Emory University School of Medicine, Renal Division, WMRB Room 338, 1639 Pierce Dr NE, Atlanta, GA 30322. E-mail jsands{at}emory.edu
Abstract
Abstract—Urea transporters have been cloned from kidney medulla (UT-A) and erythrocytes (UT-B). We determined whether UT-A proteins could be detected in heart and whether their abundance was altered by uremia or hypertension or in human heart failure. In normal rat heart, bands were detected at 56, 51, and 39 kDa. In uremic rats, the abundance of the 56-kDa protein increased 1.9-fold compared with pair-fed, sham-operated rats, whereas the 51- and 39-kDa proteins were unchanged. We also detected UT-A2 mRNA in hearts from control and uremic rats. Because uremia is accompanied by hypertension, the effects of hypertension per se were studied in uninephrectomized deoxycorticosterone acetate salt–treated rats, where the abundance of the 56-kDa protein increased 2-fold versus controls, and in angiotensin II–infused rats, where the abundance of the 56 kDa protein increased 1.8-fold versus controls. The 51- and 39-kDa proteins were unchanged in both hypertensive models. In human left ventricle myocardium, UT-A proteins were detected at 97, 56, and 51 kDa. In failing left ventricle (taken at transplant, New York Heart Association class IV), the abundance of the 56-kDa protein increased 1.4-fold, and the 51-kDa protein increased 4.3-fold versus nonfailing left ventricle (donor hearts). We conclude that (1) multiple UT-A proteins are detected in rat and human heart; (2) the 56-kDa protein is upregulated in rat heart in uremia or models of hypertension; and (3) the rat results can be extended to human heart, where 56- and 51-kDa proteins are increased during heart failure.
Key Words: urea • cardiac hypertrophy • polyamine • human heart failure • rat models
Urea is a small but highly polar molecule that has a low permeability across lipid bilayers.1 Urea transport occurs by facilitated (or carrier-mediated) pathways in kidney inner medullary collecting ducts and in erythrocytes.2 Facilitated urea transporter cDNAs have been cloned from kidney (UT-A)3 4 5 6 7 8 9 and erythrocytes (UT-B).10 11 12 13 Five different UT-A isoforms have been identified: UT-A1, UT-A2, UT-A3, UT-A4, and UT-A5. These isoforms are thought to originate from the same gene (UT-A) by alternative splicing.8 14 15
Although UT-A protein expression was originally thought to occur only in kidney, we showed that liver expresses 51- and 39-kDa UT-A proteins and that the abundance of the 51-kDa protein is significantly increased in liver from rats made uremic by 5/6 nephrectomy.16 We previously detected a 3.1-kb UT-A mRNA in rat heart by Northern analysis,8 suggesting that UT-A protein may be expressed in heart. The rationale for a cardiac urea transporter would be the same as that in liver, to dispose of urea produced in the cell. Urea production is likely increased during cardiac hypertrophy, because an increase in polyamine synthesis from ornithine is associated with cardiac hypertrophy and urea is a byproduct of the production of ornithine from arginine.17 18 19 20 21 22 23 24 25 Therefore, the goals of this study were to determine whether UT-A proteins are expressed in rat and human heart and to determine whether their abundance is altered in a variety of models: 5/6 nephrectomy rats with uremia, hypertension, and cardiac hypertrophy; deoxycorticosterone acetate (DOCA) salt–treated rats with hypertension and cardiac hypertrophy; short-term angiotensin II–infused rats with hypertension before cardiac hypertrophy; and terminal human heart failure diagnosed as dilated cardiomyopathy.
Materials and Methods
Animal Models
Animal protocols were approved by the Emory
Institutional Animal Care and Use Committee. Sprague-Dawley rats
(National Cancer Institute, Frederick, Md) were anesthetized
using intraperitoneal ketamine
(Fort Dodge Laboratories) and xylazine
(Miles Agricultural Division). For the uremia
model, rats underwent a 5/6 nephrectomy, were fed 40% protein, and
drank 1/4 normal
saline.16 26 27 28
Control rats underwent sham operation and were pair-fed. For the DOCA
model, rats underwent a right nephrectomy and a 100-mg slow-release
DOCA pellet was inserted subcutaneously through a midscapular
incision.29 Water was
replaced by 1% saline. Control rats were uninephrectomized, implanted
with a sham pellet, and given tap water to drink ad libitum. For the
angiotensin model, angiotensin II (500 ng/min
per kg) was administered by 3-day osmotic
minipump.30 Control animals
were pair-fed, and blood pressure was measured by tail
cuff.
Rat Heart
Hearts were homogenized in isolation
buffer (10 mmol/L triethanolamine, 250 mmol/L sucrose, 1
µg/mL leupeptin, and 0.1 mg/mL PMSF, pH 7.6, 0.025 to 0.1 g
tissue per mL isolation
buffer).16 31 32
Concentrated SDS was added to 1%, samples were
sheared by passage through a 28-gauge needle and centrifuged
for 15 minutes at 14 000g, and
protein was determined (DC protein assay kit,
BioRad).
Human Myocardium
Left ventricular myocardia were obtained
from 10 nonfailing and 10 terminally failing (dilated
cardiomyopathy) human hearts, which overlap with
the samples investigated
previously.33 34
The failed and nonfailed hearts were from male and female patients who
ranged in age from 33 to 59 years. Samples from left ventricle were
dissected and frozen at -80°C. Nonfailing hearts were obtained from
organ donors with brain death caused by traumatic injury. These hearts
could not be used for transplantation for reasons previously
reported.33 Left ventricle
samples from terminally failing hearts were obtained from patients
after cardiectomy during cardiac transplantation. The preoperative
diagnosis was dilated cardiomyopathy in all
patients (New York Heart Association class IV). The patients’
pretreatment consisted of diuretics and vasodilator therapy.
None received calcium channel antagonists or agonists
within 7 days of surgery or ß-adrenoreceptor agonists
within 48 hours of surgery, and none received cardiac glycoside
therapy. Patients with heart failure gave written informed consent
before operation. Drugs used for general anesthesia and
cardioplegic solutions were previously
reported.34 The experimental
protocol was approved by the local ethics review committees of both the
University of Cologne, where the heart transplantation and sample
collection took place, and the University of Southern California, where
the samples were analyzed.
Samples of heart tissue were thawed, minced, and homogenized on ice for 2 minutes with either a Polytron (Brinkmann Instruments) at a setting of 5 or a Tissuemiser (Janke and Kunkel) for 3 minutes at a thyristor setting of 55, both at 1:20 (wt:vol) in 5% sorbitol with 25 mmol/L imidazole/histidine (pH 7.4), 0.5 mmol/L Na2EDTA, and proteolytic enzyme inhibitors (1 µg/mL leupeptin, 0.5 mmol/L PMSF, and 1 mmol/L 4-aminobenzamidine dihydrochloride); protein concentrations were determined by the Lowry method.35
To determine whether UT-A protein was soluble or membrane-bound, human heart homogenate was spun at 226 000g for 3 hours. The supernatant was collected, and the pellet was resuspended in 1 mL of homogenization buffer (described above). Both the pellet, containing membrane-bound proteins, and supernatant, containing soluble proteins, were analyzed by Western blot.
Western Analysis
Total soluble proteins from rat or human hearts were
separated on 10% and 7.5%
SDS-polyacrylamide gels, respectively,
then transferred to polyvinylidine difluoride membranes
and probed with a polyclonal antibody to the C-terminus of UT-A1 that
also recognizes UT-A2 and
UT-A4.31 32
Immunoreactive proteins were visualized by enhanced chemiluminescence
(ECL, Amersham). For antibody competition
studies, the primary antibody was preincubated with the immunizing
peptide (0.1 µg/mL).31
Deglycosylation by peptide
N-glycosidase-F (PNGase F,
New England Biolabs) was performed as
described.36
Laser densitometry was used to quantitate the UT-A signal.
Results are expressed as arbitrary units per µg protein loaded.
Immunoblots of rat heart lysate were loaded with 10 µg
protein per lane based on our initial dose studies
(Figure 1
). Immunoblots of human heart lysate
were loaded with 25 µg protein per lane. Blots were quantified using
an Imaging Densitometer GS670 and Molecular Analyst software
(Bio-Rad).
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and dotted line, R=0.96), and 39-kDa bands (
and dashed line, R=0.99).
Northern Analysis
Rat UT-A4
cDNA8 was cloned into the
vector pcDNA3, which has a flanking SP6 promoter site. Templates for
GAPDH (loading control) and the molecular-weight marker probes were
linearized pTRIPLEscript plasmids: pTRI-GAPDH rat and
pTRI-MMP (Ambion). RNA
probes were synthesized by in vitro transcription incorporating
-32P-UTP (6000 Ci/mmol) and purified
through P-30 polyacrylamide gel Micro
Bio-Spin columns
(Bio-Rad). The UT-A RNA probe includes the 410
nucleotides of the 3'-end of the coding region common to
UT-A1, UT-A2, and UT-A4. Therefore, the probe detects UT-A1 (4.1-kb),
UT-A2 (3.1-kb), and UT-A4 (2.7-kb) but not UT-A3 (2.1-kb) or UT-A5
(1.4-kb).
Northern analysis of hearts from control and uremic rats was performed as described previously.37 Total RNA from individual rat hearts was isolated using TRIzol (Life Technologies), loaded (20 µg) in separate lanes, size-separated by electrophoresis on 1% agarose gels in glyoxal buffer, blotted to nylon membranes, and crosslinked with UV light. Membranes were hybridized for 2 hours at 68°C (10 cpm/mL) with UT-A probe, GAPDH probe, and then the size-marker probe. The membrane was exposed to x-ray film after each hybridization.
Reverse Transcription and Polymerase Chain
Reaction
Representative RNA samples (3 µg)
from uremic and control hearts and kidney outer medulla (control) were
reverse-transcribed to cDNA (Omniscript,
Qiagen). DNA was amplified using polymerase
chain reaction (PCR) (Advantage 2 polymerase,
Clontech) for 3 cycles (30 seconds at 95°C and
2 minutes at 70°C) and then for 32 cycles (30 seconds at 95°C and 2
minutes at 68°C). Three pairs of PCR primers were designed (Primer
Designer v4.10, Science and Educational Software). Pair 1 amplifies a
968-bp region of UT-A2 including nucleotides 1147 through
2114 (forward, 5'-CCTCTCAGTGGCATCCTCAT-3', and reverse,
5'-ACGTCGTA-GGCCTGGTACTT-3') but does not amplify UT-A4.
Pair 2 amplifies a 923-bp region of UT-A4 including
nucleotides 557 through 1480 (forward,
5'-CGATCATCGGATGAAGACAG-3', and reverse, 5'-CATGCCACCAATAGCGATAC-3').
Pair 3 amplifies a 1289-bp region of UT-A4 including
nucleotides 482 through 1771 (forward,
5'-CCTACCTGGCCTTCAAGCTC-3', and reverse, 5'-GACGTCGTAGGCCTGGTACT-3').
Neither pair 2 nor 3 amplifies UT-A2. PCR mixtures were size-separated
by electrophoresis on ethidium bromide–stained 1% agarose gels in TAE
(Tris-acetate-EDTA) buffer, and the bands were excised under UV light
and purified using QIAquick gel extraction kit
(Qiagen). Both strands of the PCR products
were fluorescently labeled (ABI Prism Dye Terminator Cycle
Sequencing Kit, Applied Biosystems), purified
with P-30 polyacrylamide columns (Micro Bio-spin,
Bio-Rad), and then sequenced using an ABI Prism
310 genetic analyzer (Applied
Biosystems).
Statistics
All data are presented as mean±SD, and n
indicates the number of rats. An unpaired Student’s
t test was used to test for
statistical significance, except for protocols in which rats were
pair-fed, where a paired Student’s
t test was
used.
Results
Normal Rats
Western analysis of rat heart tissue lysate
revealed 3 bands with molecular masses of 56, 51, and 39 kDa
(Figure 2A). The 51- and 39-kDa bands are also present in
liver.16 The 97- and 117-kDa
UT-A1
bands16 38 39
present in kidney inner medulla were not observed in rat heart. To
verify that the heart urea transporter proteins recognized by the
antibody8 31 were
not the result of some nonspecific interaction, competition studies
were performed. All 3 UT-A bands were undetectable when heart tissue
lysate was probed with antibody that had been preadsorbed with the
immunizing peptide
(Figure 2B), indicating that these proteins are
immunologically recognized as UT-A–related proteins.
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Next, heart tissue lysate was treated with PNGase F. The
56-kDa band was not present after PNGase F treatment, and a new
47-kDa band appeared
(Figure 2C
). The 51- and 39-kDa bands were present both
before and after PNGase F treatment. The disappearance of the 56-kDa
band indicates that it is N-glycosylated.
Uremic Rats
We tested whether uremia altered the abundance of any
UT-A protein in heart. Rats undergoing 5/6 nephrectomy had
significantly higher blood urea nitrogen levels (uremic, 111±49;
control, 29±11 mg/dL;
P<0.05), did not gain
weight, had significantly greater left ventricle weight, especially
when compared with body weight, and had significantly increased
systolic blood pressure
(Table).
The abundance of the 56-kDa UT-A protein was increased 1.9-fold in
hearts from uremic rats compared with the pair-fed control rats (n=8,
P<0.01,
Figure 3). There was no significant difference in the
abundance of the 51- or 39-kDa bands.
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Next, hearts from uremic rats were treated with PNGase F.
The 56-kDa band was not present after PNGase F treatment, and a new
47-kDa band appeared
(Figure 3B), similar to the results observed in untreated rat
hearts
(Figure 2C). The 51- and 39-kDa bands were present both
before and after PNGase F treatment.
UT-A mRNA
Northern analysis revealed a single 2.7-kb band
in both uremic and pair-fed control rat hearts
(Figure 4). The relative abundance of this band was unchanged
by uremia. Using reverse transcriptase (RT)-PCR, the UT-A2–specific
primer pair demonstrated the expected 968-bp product, whereas two
UT-A4–specific primer pairs did not yield products (data not
shown). All three primer pairs yielded the expected sized products
from kidney outer medulla (control). Sequencing of both DNA strands of
representative PCR products from primer pair 1
matched UT-A2 (data not shown).
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Hypertensive Rats
Because uremia is frequently associated with
hypertension, we studied the regulation of UT-A protein abundance in
hearts in other rat models associated with hypertension.
Uninephrectomized rats were treated with DOCA and given saline to drink
for 21 days.29 The absolute
heart and left ventricular weights were not different
between the DOCA-treated and control rats, but the left
ventricular weight/body weight was significantly increased
in the DOCA-treated rats compared with the control rats
(Table).
The abundance of the same 56-kDa UT-A protein was increased 2-fold in
the DOCA-treated rats compared with control rats (n=7,
P<0.005,
Figure 5). There was no significant difference in the
abundance of the 51- or 39-kDa bands.
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Next, we studied a more acute model of hypertension,
administering angiotensin II for 3 days.
Angiotensin II treatment significantly increased
systolic blood pressure (n=5,
P<0.005,
Table)
and plasma aldosterone levels (angiotensin II
rats, 131±74; control rats, 9±12 ng/dL; n=4,
P<0.01). There was no
significant difference in heart or left ventricle weight or left
ventricle/body weight between rats receiving angiotensin II
and control rats
(Table).
However, the abundance of the same 56-kDa UT-A protein was increased
1.8-fold in the angiotensin II–treated rats compared with
control rats (n=9, P<0.05,
Figure 6). There was no significant difference in the
abundance of the 51- or 39-kDa bands.
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Human Heart Failure
To assess whether the changes that we observed in UT-A
protein expression in the experimental rat models could be extended to
pathological changes in human heart, we determined whether UT-A
proteins were present in human heart and whether their abundance
changed in heart failure. Western analysis of nonfailing human
heart tissue lysate revealed immunoreactive bands with apparent
molecular masses of 97, 56, and 51 and a fainter band
corresponding to the mobility of the rat 39-kDa protein
(Figure 7A). Only the 97-, 56-, and 51-kDa bands were
detected in every sample. The 97-kDa band is analogous to the smaller
UT-A1 protein detected in rat kidney inner medulla
(Figure 2A); the 56- and 51-kDa bands are analogous to the
UT-A proteins detected in rat heart.
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Subcellular fractionation showed that the immunoreactive
UT-A bands were present in the pellet
(Figure 7B), indicating that they were in the membrane
fraction. No bands were detected in the supernatant (data not shown),
indicating that the UT-A bands were not soluble proteins.
In the terminally failed human hearts, the abundance of the
56-kDa UT-A1 protein increased 1.4-fold
(P<0.05), and the 51-kDa UT-A
protein increased 4.3-fold
(P<0.005) compared with
nonfailed human hearts
(Figures 7C and 7D). The 97-kDa UT-A protein increased
1.37-fold, although this change did not achieve statistical
significance.
Discussion
The major findings in this study are that rat heart expresses 3 UT-A proteins with molecular masses of 56-, 51-, and 39-kDa and that human heart expresses 4 UT-A proteins with molecular masses of 97-, 56-, 51-, and 39-kDa. In rat, the abundance of the 56-kDa UT-A glycoprotein is significantly increased by uremia, accompanied by hypertension and cardiac hypertrophy. The 56-kDa UT-A protein is also increased in nonuremic DOCA/salt hypertension accompanied by cardiac hypertrophy and in short-term hypertension induced by a 3-day infusion of angiotensin II (before cardiac hypertrophy is apparent). In terminally failing human hearts, diagnosed as dilated cardiomyopathy (New York Heart Association class IV), there are also significant increases in the abundance of UT-A proteins at 56 and 51 kDa.
Extrarenal Expression of UT-A
We showed that UT-A proteins are expressed in rat liver
and brain.16 In this study,
we identified multiple UT-A protein bands in rat and human heart,
although by Northern analysis we detect only a single message.
Multiple proteins arising from a single message are not without
precedent; the cardiac sarcolemmal
Na+-Ca2+
exchanger is a single mRNA that gives rise to 3
proteins.40 However, given
the complexity of the UT-A
gene,14 15 we
cannot exclude the possibility that other, currently unidentified UT-A
isoforms are also expressed in heart.
Previously, we observed a 3.1-kb mRNA band in rat heart that corresponds to the size of UT-A2,8 whereas You et al4 did not detect any UT-A mRNA in rabbit heart. In this study, we detected a 2.7-kb transcript, and the DNA sequence of the RT-PCR product-matched UT-A2.8 A smaller UT-A2 transcript named UT-A2b (2.5 kb), which has the same coding region as UT-A2 but a shorter 3' untranslated region, is expressed in kidney inner medulla.41 Because the PCR product that we sequenced represents almost the entire coding region of UT-A2 (968 of 1190 bp), the small difference in mRNA size on Northern analysis may indicate that the heart transcript results from differences in the untranslated regions of UT-A2. However, to prove this would require cloning the UT-A2 transcript from heart.
Although we cannot definitively assign a UT-A isoform number to each of the UT-A protein bands, the 97-kDa band observed in the human heart migrates at the same mobility as one of the UT-A1 glycoproteins in kidney inner medulla,42 and the 56-kDa heart band migrates at the same molecular mass as UT-A2 in rat kidney outer medulla.43 The 51- and 39-kDa heart bands comigrate with the UT-A bands identified in liver.16 On treatment with PNGase F, the 56-kDa band disappeared and a 47-kDa band appeared in hearts from either control or uremic rats. The 51- and 39-kDa bands do not seem to be changed by PNGase F treatment, similar to the result of PNGase F treatment in liver.16 These findings suggest that the 56-kDa protein is being deglycosylated to a 47-kDa protein. However, we cannot exclude the possibility that the 56- and 51-kDa proteins are being deglycosylated to 51- and 47-kDa proteins, respectively. All heart samples were kept on ice, and protease inhibitors were added in an attempt to avoid any proteolysis. Nevertheless, we cannot exclude the possibility that some of the heart bands are proteolytic products.
Possible Physiological Role
for UT-A in Heart
Whereas the presence of UT-A urea transporter proteins
in kidney and liver is logical considering the role of urea in the
urine concentrating mechanism and the fact that liver is the principal
site of ureagenesis, the function of a urea transporter in heart is not
as obvious. In liver, urea production occurs primarily from
arginine in the urea cycle. In extrahepatic tissue, urea can also be
produced as a byproduct of ornithine synthesis from arginine via
arginase in the first step of the polyamine synthesis
pathway.23 24 44
Spermatogenesis is associated with an increase in urea
production by the polyamine pathway, and urea transporter
proteins are present in the Sertoli cells of rodent seminiferous
tubules.9 11 The
polyamine pathway is present in heart, and polyamine
production via the rate-controlling enzyme, ornithine
decarboxylase, increases in conditions associated with cardiac
hypertrophy.17 18 19 20 21 22 23 24 25
The increase in polyamine synthesis may be a critical contributor to
cardiac hypertrophy, because ornithine decarboxylase
inhibitors protect the heart from becoming hypertrophic
during aortic coarctation or during treatment with a ß-adrenergic
agonist in vivo or in cultured
cardiomyocytes.18 45 46
The hypertrophic response is also dependent on autocrine stimulation
with transforming growth
factor-ß.45 However, the
dependence of cardiac hypertrophy on polyamine
production has been questioned in transgenic mice that
overexpress an inhibitor of ornithine decarboxylase,
antizyme-1, because ß-adrenergic agonist-induced cardiac
hypertrophy occurs without an increase in polyamine
synthesis.47
Whether or not the induction of polyamine synthesis is
critical for cardiac hypertrophy, there is agreement that
it is activated in association with cardiac
hypertrophy. In the present study, we found a
significant increase in the left ventricle/body weight in both uremic
rats and the DOCA salt–treated rats
(Table),
which is evidence for the presence of left ventricular
hypertrophy. Although the left ventricle/body weight was
not increased in the angiotensin II–treated rats, these
rats received angiotensin II for only 3 days, which may not
have been long enough to detect significant cardiac
hypertrophy. In an earlier study of angiotensin
II–infused rats, increased polyamine synthesis in the heart was
detected within hours, whereas measurable levels of cardiac
hypertrophy were not detected until angiotensin
II had been infused for several
days.21
Relevant to the human heart, a study of patients with chronic heart failure established that the plasma urea level was an independent prognostic factor of mortality.48 Half of the patients with elevated plasma urea also had elevated creatinine, evidence of decreased renal function, but half did not, perhaps evidence of increased catabolism, a stimulus that might warrant increased urea transporter abundance in tissues such as heart.
In the human heart samples used in this study, we previously
studied how heart failure affects the levels of sodium pump isoforms
and Na+-Ca2+
exchanger protein levels in the left ventricle. Protein levels of
Na+,K+-ATPase
1, 3, and
ß1 (but not 2) were
significantly reduced to between 60% and 70% of control, as were
Na+,K+-ATPase
activity and ouabain binding, whereas levels of
Na+-Ca2+
exchanger and calsequestrin remained
unchanged.34 Thus, membrane
transport proteins seem to display unique patterns of change in human
heart failure, indicating that the changes are not secondary to changes
in the cell membrane to volume relationship. We speculate that
upregulation of the UT-A proteins in the hearts of uremic or
hypertensive rats and in human dilated
cardiomyopathy may be important for urea exit in
conditions where urea production is
increased.
Summary
This study shows the expression of UT-A proteins in rat
and human heart lysate. In rat, the abundance of the 56-kDa UT-A
protein is increased in conditions (uremia and hypertension) that
predispose to left ventricular hypertrophy and
suggest the hypothesis that upregulation of the 56-kDa UT-A protein in
the heart of uremic rats may be related to hypertension. This
observation was reexamined in a set of human hearts diagnosed as
dilated cardiomyopathy, where it was found that the
abundance of the 56- and 51-kDa UT-A proteins was significantly
increased compared with their expression in nonfailing
hearts.
Acknowledgments
This work was supported by National Institutes of Health grants R01-DK41707 and P01-DK50268 (to J.M.S.) and by a Grant-in-Aid from the American Heart Association Western States Affiliate (to A.A.M.). The authors thank Dr Robert H.G. Schwinger and his colleagues (Klinik III for Innere Medizen, University of Cologne, Germany) for providing myocardial tissue samples, Dr David G. Harrison (Emory University, Atlanta, Ga) for providing hearts from DOCA-treated rats, and Dr William E. Mitch (Emory University) for critically reading this manuscript.
Footnotes
Original received January 30, 2001; revision received May 18, 2001; accepted May 18, 2001.
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