Vol. 277, Issue 4, H1418-H1428, October 1999
Regional alterations in SR
Ca2+-ATPase, phospholamban,
and HSP-70 expression in chronic hibernating myocardium
James A.
Fallavollita,
Saji
Jacob,
Rebeccah F.
Young, and
John M.
Canty Jr.
Department of Veterans Affairs, Western New York Health Care
System, and Departments of Medicine and Physiology, University at
Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New
York 14214
 |
ABSTRACT |
We sought to identify mechanisms for chronic
dysfunction in hibernating myocardium. Pigs were instrumented with a
left anterior descending artery stenosis for 3 mo. Angiography
demonstrated high-grade stenoses and hibernating myocardium with
1) severe anterior hypokinesis
(P < 0.001 vs. shams),
2) reduced subendocardial perfusion
[0.73 ± 0.05 (SE) vs. 1.01 ± 0.06 ml · min
1 · g
1
in normal, P < 0.001], and
3) critically reduced adenosine flow (1.0 ± 0.17 vs. 3.84 ± 0.26 ml · min
1 · g
1
in normal, P < 0.001). Histology did
not reveal necrosis. Northern blot analysis of hibernating myocardium
demonstrated regional downregulation in mRNAs for sarcoplasmic
reticulum (SR) proteins phospholamban (0.76 ± 0.08 vs. 1.07 ± 0.06, P < 0.02) and SR
Ca2+-ATPase (0.83 ± 0.06 vs.
1.02 ± 0.06, P < 0.05)
with no change in calsequestrin (1.08 ± 0.06 vs. 0.96 ± 0.05, P = not significant). Heat shock protein (HSP)-70 mRNA was regionally induced in hibernating myocardium (2.4 ± 0.3 vs. 1.0 ± 0.11, P < 0.01). Directionally similar
changes were confirmed by Western blot analysis of respective proteins.
Our results indicate that hibernating myocardium exhibits a molecular
phenotype that on a regional basis is similar to end-stage ischemic
cardiomyopathy. This supports the hypothesis that SR dysfunction from
reversible ischemia may be an early defect in the progression
of left ventricular dysfunction.
coronary flow; myocardial ischemia; calcium regulatory
proteins; sarcoplasmic reticulum
 |
INTRODUCTION |
ALTHOUGH ISCHEMIC cardiomyopathy is the leading
etiological cause of congestive heart failure (15), the mechanisms
through which chronic coronary artery disease progresses to
decompensated left ventricular dysfunction are complex and poorly
understood (33). Severe coronary artery disease and a history of
symptomatic ischemia are present in the majority of patients,
but the role of reduced coronary flow reserve in the progression of
contractile dysfunction has not been resolved. Heart failure in
coronary disease can arise from structural alterations consisting of
myocyte necrosis and replacement fibrosis (3, 38), but contractile
dysfunction due to abnormalities of intracellular calcium and
alterations in myocyte function have also been implicated to play an
important role (31). Explanted hearts from patients with advanced
ischemic cardiomyopathy have reductions in the expression of some, but not all, of the sarcoplasmic reticulum (SR) calcium handling proteins (29), and in vitro studies demonstrate attenuated force-interval relationships in explanted cardiac muscle (34). This suggests that the
contractile abnormalities may be due to SR dysfunction. Nevertheless,
whether SR dysfunction arises secondary to global systemic hemodynamic
changes or neurohormonal activation associated with advanced heart
failure, or from repetitive myocardial ischemia due to
extensive coronary artery disease has not been studied because it is
difficult to dissociate these factors clinically or in experimental
animal models of heart failure.
Chronically dysfunctional myocardium that is viable or
"hibernating" is frequently found in patients with severe
coronary artery disease and collateral-dependent myocardium (2, 45). Although this can occur in the absence of heart failure, it can also
contribute to the progression of left ventricular dysfunction to
clinical heart failure (15). The mechanisms responsible for chronic
contractile dysfunction in hibernating myocardium are unclear, as are
their relation to global alterations in advanced ischemic
cardiomyopathy. One possibility is that the chronic alterations in SR
proteins found in advanced heart failure are actually an early
abnormality secondary to an intrinsic myocardial adaptation from
intermittent ischemia. This may precede the development of structural fibrosis, neurohormonal activation, and global systemic abnormalities found in end-stage heart failure.
To test this hypothesis, we used a recently developed porcine model of
regional, viable, dysfunctional myocardium that has all of the features
of hibernating myocardium in humans, including regional reductions in
resting flow and function and increased 18F-2-deoxyglucose uptake (12).
The direct effects of a chronic reduction in regional coronary flow
reserve could be studied because these animals do not develop systemic
changes seen in heart failure. Our first aim was to study whether there
was a regional downregulation in selected SR proteins that was similar
to heart failure and to determine how this is related to coronary
physiology in the intact animal. The second aim was to examine whether
a chronic stenosis could be associated with the induction of mechanisms that protected myocytes from ischemia. To do this, we initially chose to examine heat shock protein (HSP)-70 that has been shown to be
induced following brief total coronary occlusions and associated with
protection against stunning and infarction (27, 43). The results
indicate that hibernating myocardium exhibits a molecular phenotype
that, while regional, is similar to global changes in ischemic
cardiomyopathy. Thus SR dysfunction due to reversible ischemia
may occur as an early defect in the progression of left ventricular
dysfunction that precedes the development of the structural abnormalities seen in ischemic cardiomyopathy.
 |
METHODS |
All experimental procedures and protocols conformed to Institutional
Guidelines for the Care and Use of Animals in Research. Studies were
conducted in 24 hibernating animals that had a proximal left anterior
descending artery (LAD) occlusion and 13 sham-instrumented controls.
Terminal hemodynamic and coronary flow studies were performed in 22 hibernating animals (4 used for protein analysis and 18 used for RNA
assessment) and 11 sham controls. The remaining four animals (2 hibernating and 2 sham) were killed without physiological studies to
confirm that acute instrumentation had no affect on mRNA levels.
Chronic surgical preparation.
The surgical preparation has been previously described (12). Briefly,
juvenile pigs were fasted overnight, premedicated with a Telazol (50 mg/ml tiletamine and 50 mg/ml zolazepam)-ketamine (100 mg/ml) mixture
(0.037 ml/kg im), and given prophylactic antibiotics (50 mg/kg iv
cephalothin and 5 mg/kg im gentamicin). After endotracheal intubation a
surgical plane of anesthesia was maintained with a halothane
(0.5-2%) and oxygen (balance) mixture. A thoracotomy was
performed in the fourth left intercostal space, and the proximal LAD
was dissected free and instrumented with a Delran occluder [average ID 1.7 ± 0.1 (SE) mm]. In hibernating animals,
the artery was secured within the occluder by a silk ligature or
umbilical tape. In sham animals, the groove of the occluder was left
open to circumvent the formation of a significant stenosis. After
surgery was complete, the chest incision was closed in layers, the
intercostal nerves were infiltrated with 2% lidocaine for analgesia,
and the pneumothorax was evacuated. A postoperative dose of antibiotics was repeated, and an analgesic (0.025 mg/kg im butorphanol) was given
and repeated as required to alleviate pain. Animals were fully
recovered within 24 h. They were group housed and fed a diet of
Pro-Lean (Agway, Akron, NY) ad libitum.
Hemodynamic studies in hibernating and sham-instrumented animals.
Animals were brought back to the laboratory for physiological studies
and tissue harvesting after ~3-4 mo. They were fasted overnight,
and anesthesia was induced with a Telazol-xylazine (100 mg/ml) mixture
(0.022 ml/kg im). After tracheal intubation, a surgical plane of
anesthesia was maintained using a halothane (0.5-2%) and oxygen
(balance) mixture supplemented with intramuscular doses of
Telazol-xylazine (0.011 ml/kg) as required. The left carotid artery was
instrumented with a 7-Fr introducer for coronary angiography. The right
femoral artery was dissected free and used to pass an 8-Fr pigtail
catheter retrogradely into the left ventricle using fluoroscopic
guidance. A short catheter was placed into the left
femoral artery to withdraw blood for microspheres using the reference
sampling technique. Catheters were placed in the femoral veins to
administer fluids (normal saline) and pharmacological agents. The
animals were heparinized (10,000 U iv), and hemodynamics were allowed
to equilibrate for at least 30 min before the protocol was begun.
After equilibration, colored microspheres were injected into the left
ventricle to assess regional perfusion under resting conditions
followed by contrast left ventriculography with hand injections of
10-20 ml of iohexol (Winthrop Pharmaceuticals, New York, NY).
After resting measurements, an infusion of epinephrine was started and
titrated so that heart rate increased to ~120-140 beats/min
(0.27 ± 0.03 µg · kg
1 · min
1
iv). Once hemodynamics reached a steady state (~10 min), a second microsphere flow measurement was performed followed by a left ventriculogram. After the epinephrine was stopped and hemodynamics returned to baseline, pharmacological vasodilation was produced to
assess regional coronary flow reserve using intravenous adenosine (0.9 mg · kg
1 · min
1
iv). Because this dose of adenosine was accompanied by arterial hypotension without a reflex tachycardia in pigs, a simultaneous infusion of phenylephrine (5.5 ± 0.6 µg · kg
1 · min
1
iv) was started and titrated to restore systolic blood pressure to
control levels. After the protocol was completed, selective coronary
angiography was performed as previously described (12).
After angiography, the animals were deeply anesthetized and the heart
was rapidly excised and weighed. Samples from the anterior freewall and
septum that were immediately adjacent to the LAD and approximately
midway between the occluder and apex were taken for microsphere flow
analysis (duplicate samples) and RNA or protein isolation. Similar
samples were taken from the normal region midway between the base and
apex at the junction of the posterior freewall and septum. Our previous
studies demonstrated that these regions systematically represented
tissue from the central regions of LAD and normally perfused
myocardium. All samples were cut into three transmural layers of
approximately equal thickness. Samples for RNA isolation were handled
with precautions to prevent RNA degradation, flash frozen in liquid
nitrogen, and stored at
80°C until analyzed.
Microsphere flow and angiographic measurements.
Regional perfusion was assessed with colored microspheres (15- µm
diameter, Dye-Trak, Triton, San Diego, CA) using previously published
techniques (12). Briefly, microspheres suspended in saline with
thimerosal (0.01%) and Tween 80 (0.01%) were sonicated and vortex
agitated before withdrawal. Approximately 3-5 million microspheres
(yellow, red, white, or blue) were administered as a left ventricular
bolus after an arterial reference sample was started from the femoral
artery. At the end of the study, myocardial samples were weighed,
digested in 4 M KOH with 2% Tween 80, and processed using previously
published techniques (20). The dyes were eluted from the microspheres
using a measured volume of dimethylformamide and aliquots placed in a
multiple wavelength spectrophotometer (model U-2000, Hitachi, Tokyo,
Japan). Absorbance was measured at the principal absorbance peak of
each pure color dye and corrected for overlapping spectra using a
matrix inversion technique (18, 20). Regional myocardial perfusion was
calculated using the absorbance and flow rate of the arterial reference
sample and myocardial absorbance per unit sample weight (20).
Magnified coronary angiograms were used to quantify the percent
stenosis. End-systolic and end-diastolic images of the left ventricle
were traced by two observers. Global ejection fraction was calculated
from digitized tracings using the area length method (37), and the
measurements from each observer were averaged. Regional wall motion in
the anteroapical wall was assessed using the following scoring system:
3, normal; 2, mild hypokinesis; 1, severe hypokinesis; and 0, akinesis.
Dyskinesis was not present under any condition. Ventriculography could
not be performed in three animals from the hibernating group, and
coronary angiography could not be completed in five animals from the
hibernating group due to technical limitations.
RNA isolation and Northern blot analysis.
Total myocardial RNA was isolated from subendocardial samples (inner
one-third) of the central LAD and normal remote regions by guanidinium
thiocyanate-phenol chloroform extraction (9). Because large variations
in HSP-70 expression were found, we also examined its distribution in
mid- and subepicardial samples. We simultaneously electrophoresed
20-µg aliquots of RNA on 0.8% agarose formaldehyde gels. These were
vacuum transferred onto nylon membranes and baked in a vacuum oven at
80°C. We hybridized Northern blots with a 2.3-kb probe for human
HSP-70 (47) [American Type Culture Collection (ATCC), Rockville,
MD]. Control for RNA loading was confirmed and quantified using a
1.2-kb probe to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ATCC)
(44). The remaining probes for
Ca2+-ATPase, calsequestrin,
calmodulin, phospholamban,
-myosin heavy chain, and the ryanodine
receptor were synthesized by PCR. First-strand cDNA was synthesized by
reverse transcribing myocardial mRNA with M-MLV Reverse Transcriptase
at 37°C for 1 h. This was subsequently amplified by PCR using
primers for the specific nucleotide sequence coding for each protein.
After an initial denaturation for 5 min at 94°C, samples were
cycled 25 times at 94°C for 30 s, at 45°C for 1 min, and at
72°C for 1 min. This was followed by a final 7-min extension at
72°C. The following pairs of oligonucleotide primers were used to
amplify probes from porcine myocardial cDNA with the exception of SR
Ca2+-ATPase, for which rat
myocardial cDNA was used: 1) rat SR
Ca2+-ATPase (24), sense primer
5'-TTGGCTTGGTTCGAAGAAGG-3' (+223 to +242 nt) and antisense
primer 5'-CCAAGAGCCACCATGAACTG-3' (+864 to +845 nt) to
produce a 642-bp fragment; 2)
calsequestrin (40), sense primer 5'-AAGCTTGCCAAGAAGCTGGG-3'
(+301 to +320 nt) and antisense primer
5'-GCAAAGGCCACAATGTGGAT-3' (+821 to +802 nt) to yield a
521-bp product; 3) calmodulin (13),
sense primer 5'-AGGAGTTGGGGACAGTGATG-3' (+192 to +211 nt)
and antisense primer 5'-ATGTCAGCCTCCCTGATCAT-3' (+492 to
+473 nt) to produce a 301-bp segment;
4) phospholamban (46), sense primer
5'-TCAGCTTTCTCTTGACGGCT-3' (
52 to
33 nt) and
antisense primer 5'-ACCCCTAGTTCATCCTCAGA-3' (+474 to +455
nt) to produce a 526-bp fragment; 5)
-myosin heavy chain (21), sense primer
5'-TGAAGGAGAACATCGCCATC-3' (+788 to +807 nt) and antisense
primer 5'-GTAGGTGAGCTCCTTGATGC-3' (+1344 to +1325 nt) to
yield a 557-bp product; and 6) the
cardiac ryanodine receptor (RYR2) (22), sense primer
5'-GGAAATCCATTCTGAATTCT-3' (+21 to +40 nt) and antisense
primer 5'-GCAGTCACAAACGGCTCGGT-3' (+488 to +507 nt) to
produce a 487-bp segment. The amplified cDNA products were inserted
into a vector [PCR-TRAP kit from GenHunter (Boston, MA) or TA
Cloning kit from Invitrogen (San Diego, CA)] and transformed into
Escherichia coli. HSP-70 and GAPDH
probes were labeled by random priming. All other probes were labeled by
PCR in the presence of
[
-32P]dCTP (30 cycles at 94°C, 30 s; 50°C, 1 min; 72°C, 1 min).
Beta emissions were quantified on a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA) after overnight exposure. Values presented are in
arbitrary densitometric units normalized to the GAPDH value for the
sample to control for variations in loading. Average ratios for the LAD
samples are compared with the normal remote regions of the same animals.
Protein isolation and Western blot analysis.
To confirm that mRNA alterations reflected steady-state changes in
protein levels, subendocardial samples were obtained from four animals
with regional reductions in resting perfusion that underwent identical
instrumentation. Protein was isolated from flash-frozen samples using
an extraction buffer containing 20 mM Tris (pH 7.4), 50 mM NaCl, 1 mM
EDTA (pH 8), 1 µg/ml pepstatin, 0.5 µg/ml leupeptin, 5 mM
-mercaptoethanol, 0.2 mM sodium vanadate, 10% SDS, and 0.2 mM
phenylmethylsulfonyl fluoride. Protein concentration for each sample
was measured in triplicate using the Lowry method (25). Aliquots of
protein (50 µg/lane for SR
Ca2+-ATPase, 75 µg/lane
calsequestrin and phospholamban, and 80 µg/lane for HSP-70) were
electrophoresed and separated on 10 or 14% SDS-polyacrylamide gels.
They were transferred to a nitrocellulose membrane (HSP-70, Protran,
Schleicher and Schuell, Keene, NH) or a polyvinylidene difluoride
membrane (other proteins, Immobilon-P, Millipore, Bedford, MA) and
blocked in 3% nonfat dry milk in PBS. After the membrane was rinsed,
it was incubated overnight with the primary antibody [SR
Ca2+-ATPase anti-canine mouse
monoclonal antibody (32), 1:1,500 dilution (MA3-919, Affinity
Bioreagents, Golden, CO)]; phospholamban anti-canine mouse
monoclonal antibody, 1:1,000 dilution (05-205, Upstate
Biotechnology, Lake Placid, NY); calsequestrin anti-canine rabbit
polyclonal antibody, 1:800 dilution (06-382, Upstate
Biotechnology); and HSP-70 anti-human mouse monoclonal antibody, which
recognizes both the constitutive (73 kDa) and inducible (72 kDa) forms
of HSP-70, 1:5,000 dilution (SPA-820, Stress Gen Biotechnology,
Victoria, BC). After being rinsed in PBS, membranes were incubated with peroxidase-labeled goat antibodies to IgG. Bands were visualized with
3,3',5,5'-tetramethylbenzidine (TMB) membrane peroxidase substrate (Kirkegaard and Perry, Gaithersburg, MD) and quantified on a
Laser Densitometer (Bio-Rad, Melville, NY). Linearity of density and protein loading for the SR
Ca2+-ATPase was demonstrated over
a range of 20-60 mg of total protein, between 25 and 100 mg of
total protein for phospholamban, and between 40 and 140 mg total
protein for calsequestrin and HSP-70.
Histology.
Myocardial samples adjacent to those taken for RNA and protein analyses
were immersed in Z-fix (Anatech, Battle Creek, MI). Thin sections were
stained with Masson's trichrome stain to delineate connective tissue
and fibroblast staining from normal myocytes. Percent connective tissue
staining was quantified with standard point counting techniques using a
121-point grid at a magnification of ×200 as we have previously
described (12). Two full-thickness regions were analyzed in each sample
and selected to represent the area of greatest and least connective
tissue staining in the sample. At least 30 fields were quantified and
averaged for each sample. Connective tissue staining was expressed as
a percentage of the total. Perivascular connective tissue
staining and endocardial staining were included in the reported results.
Data analysis.
All data are presented as means ± SE. Differences between sham and
hibernating animals were determined using group
t-tests for similar parameters and
interventions. Measurements in the LAD region were compared with
corresponding measurements in the normal region using paired
t-tests. Differences between rest and pharmacological interventions were assessed using an ANOVA followed by
paired t-tests and the Bonferroni
correction for multiple comparisons. P < 0.05 level was considered significant.
 |
RESULTS |
Myocardial perfusion and hemodynamics in hibernating versus sham
controls.
All animals were in good health at the time of study. Data for the
entire hibernating group (n = 22) are
summarized below. Body weight increased from 9.2 ± 0.4 kg at the
time of instrumentation to 83.0 ± 3.4 kg at the time of study
(average growth interval 102 ± 3 days). Left ventricular mass
averaged 217 ± 9 g, and mass-to-body weight ratio averaged 2.63 ± 0.07 g/kg. Arterial blood gases immediately before resting
measurements of myocardial perfusion averaged pH 7.40 ± 0.01, PCO2 38 ± 1 mmHg, and
PO2 477 ± 28 mmHg. Hematocrit
averaged 33 ± 1%. Under resting conditions, heart rate averaged 83 ± 3 beats/min, and systolic blood pressure averaged 121 ± 4 mmHg. Resting subendocardial perfusion averaged 0.73 ± 0.05 ml · min
1 · g
1
in LAD regions and 1.01 ± 0.06 ml · min
1 · g
1
in normal remote regions (P < 0.001). Corresponding full-thickness values were 0.80 ± 0.05 and 0.92 ± 0.06 ml · min
1 · g
1
(P < 0.001). During adenosine
vasodilation, subendocardial flow in the LAD region did not increase
significantly over rest values [1.0 ± 0.17 ml · min
1 · g
1,
P = not significant (ns) vs.
rest], but in normal remote regions it increased to 3.84 ± 0.26 ml · min
1 · g
1
(P < 0.001 vs. rest). Corresponding
full thickness values were 1.42 ± 0.16 ml · min
1 · g
1
in the LAD region (P < 0.01 vs.
rest) and 4.33 ± 0.24 ml · min
1 · g
1
in the remote normal zone (P < 0.001 vs. rest).
A representative left coronary angiogram (Fig.
1A) and
corresponding end-systolic and end-diastolic tracings of the resting left ventriculogram (Fig. 1B) in the
left lateral projection from a hibernating animal are shown. Analysis
of the left ventriculograms revealed a resting ejection fraction of 52 ± 3% in hibernating animals vs. 56 ± 3% in shams
(P = ns). Although there was no
difference in global function, regional anteroapical wall motion was
severely depressed (wall motion score 1.0 ± 0.2 in hibernating
animals vs. 2.1 ± 0.2 in shams, P < 0.001). After epinephrine, regional wall motion increased in
hibernating animals to 1.3 ± 0.2 (P < 0.05) with a
borderline increase in global ejection fraction to 57 ± 5%
(P = 0.08). Hibernating animals had
severe proximal stenoses or total LAD occlusion with angiographic
collaterals (mean stenosis severity for the group was 97 ± 2%). In
the sham group (n = 11) the average
LAD diameter stenosis was 43 ± 4%, reflecting the fact that the
artery was able to remodel and expand out of the open occluder, whereas
this was prevented by the ligature or umbilical tape in the hibernating
group. There were no angiographic collaterals visible in the sham
group. Thus hibernating animals were characterized by severe
hypokinesis of the anteroapical wall on ventriculography and severe
proximal stenosis or total occlusion of the LAD with angiographically
visible collaterals opacifying the distal LAD.


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Fig. 1.
Coronary angiogram and tracings of a resting left ventriculogram from a
hibernating pig. Angiogram and tracings of left ventriculogram are in
the left lateral projection. Coronary angiography in animals in the
hibernating group (A) usually showed
total occlusion of proximal left anterior descending artery (LAD;
arrow), which was associated with brisk opacification of distal LAD by
angiographically visible collaterals. Ventriculography
(B) showed severe hypokinesis
or akinesis of anteroapical wall of left ventricle. In contrast,
sham-instrumented animals had mild stenosis of the proximal LAD and
preserved anteroapical wall motion. Solid line, end-diastolic (ED)
tracing; dotted line, end-systolic (ES) tracing; LC, left
circumflex artery.
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To determine whether relative reductions in flow were due to increased
demand in remote zones in the face of a large dysfunctional region,
measurements of hemodynamics and transmural myocardial perfusion were
performed in a subgroup of concurrent hibernating (n = 10) and sham-instrumented
(n = 10) pigs (Tables
1 and 2). Resting flow was identical in normal remote myocardium of hibernating pigs and sham-instrumented controls, indicating that relative reductions in flow were not due to increased metabolic demands and
contractility. During metabolic stimulation with epinephrine, subendocardial flow in the LAD region did not increase significantly (0.65 ± 0.09 to 0.66 ± 0.16 ml · min
1 · g
1),
whereas there was no regional variation in sham-instrumented controls.
Histological analysis showed a subtle, but generalized, increase in the
usual connective tissue staining in the LAD region from both
hibernating and sham-instrumented pigs. The extent of connective tissue
staining (including perivascular tissue) assessed by point counting
averaged 5.7 ± 0.6% in the LAD region of hibernating animals
versus 3.2 ± 0.3% in the LAD region of shams
(P < 0.05). Values were also lower
in the normal regions from hibernating animals and averaged 3.6 ± 0.3% (P < 0.01 vs. corresponding
LAD values).
Alterations in SR
Ca2+-ATPase and
phospholamban mRNA in hibernating myocardium.
Figure 2 is a representative Northern blot
for paired samples from four hearts comparing mRNA levels in
dysfunctional hibernating regions with their corresponding expression
in normal remote regions from the same heart. Figure 3
summarizes the results of representative SR mRNAs from paired samples
from all of the hibernating animals that were studied. Data were
normalized to the GAPDH expression in each sample, which did not vary
between regions (subendocardial GAPDH 2.96 ± 0.23 densitometric
units in hibernating LAD regions versus 3.03 ± 0.24 densitometric
units in normal remote regions, P = ns). Although there was variability among animals, we found highly
significant reductions in the level of mRNA for phospholamban and the
SR Ca2+-ATPase in hibernating LAD
regions compared with normal regions, whereas mRNA for the SR calcium
binding protein calsequestrin was unchanged. The relative changes in
mRNA (LAD/normal) were also reduced in comparison to sham-instrumented
controls as summarized in Fig. 4 (phospholamban 0.76 ± 0.08 in hibernating vs. 1.07 ± 0.06 in shams,
P < 0.02; SR
Ca2+-ATPase 0.83 ± 0.06 in
hibernating vs. 1.02 ± 0.06 in shams,
P < 0.05; calsequestrin 1.08 ± 0.06 in hibernating vs. 0.96 ± 0.05 in shams,
P = ns). Analysis of the ryanodine
receptor mRNA was performed only in hibernating animals. The expression
of RYR2 relative to GAPDH was reduced in hibernating compared with
normal remote regions (LAD RYR2/GAPDH 0.12 ± 0.01 vs. 0.15 ± 0.01 in normal regions, n = 17, P < 0.05; LAD/normal 0.85 ± 0.07). These data indicate that there were significant reductions in
the mRNAs for the SR calcium uptake and release proteins, whereas the
constancy of mRNA for calsequestrin suggests that this did not reflect
a nonspecific alteration in the expression of all SR proteins.

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Fig. 2.
Representative Northern blot of sarcoplasmic reticulum (SR) calcium
handling protein mRNA from subendocardium in normal (Nl) and
hibernating (H) regions. Each pair of lanes depicts subendocardial mRNA
from individual animal. Bottom panel
shows that there was relatively uniform loading and no change in
expression of control probe for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). In contrast, there was reduction in expression
of phospholamban (both upper and lower bands) and
SR Ca2+-ATPase in hibernating LAD regions. Although
variable, the degree to which phospholamban was reduced generally
paralleled that for the SR
Ca2+-ATPase in individual
animals.
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Fig. 3.
Paired comparison of subendocardial SR gene expression in hibernating
LAD vs. normal remote regions (n = 20). A: phospholamban. B: SR Ca2+-ATPase.
C: calsequestrin. Although there was variation among individual
animals, there were significant reductions in the mRNA levels for
phospholamban and the SR
Ca2+-ATPase in hibernating vs.
normally perfused regions. In contrast, expression of calsequestrin
remained unchanged. Solid symbols represent group averages (means ± SE).
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Fig. 4.
Relative induction of subendocardial SR
Ca2+ regulatory protein mRNA in
hibernating (n = 20) vs.
sham-instrumented animals (n = 10).
Relative mRNA induction is expressed as ratio of LAD to normal zone
mRNA normalized to respective expression of GAPDH in each region.
Sham-instrumented animals demonstrated no regional alteration in
expression of phospholamban, SR
Ca2+-ATPase, or calsequestrin.
Hibernating animals demonstrated significant regional reductions in SR
Ca2+-ATPase and phospholamban mRNA
in comparison to shams, whereas expression of calsequestrin mRNA was
unaltered. Pattern of gene expression demonstrated on regional basis is
similar to global changes found in advanced congestive heart failure.
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Transmural induction of HSP-70 mRNA in hibernating animals.
We found a graded induction of mRNA for HSP-70 across the wall of the
LAD region of hibernating animals. Figure
5 depicts the
results of a representative gel from an individual animal with
hibernating myocardium. There was a low uniform HSP-70 mRNA expression
in all layers of the normally perfused region that was similar to that
seen in sham-instrumented animals. In contrast, there was an induction
of HSP-70 mRNA in hibernating LAD regions that was greatest in the
subendocardium and least in the subepicardium. Figure
6 compares the induction of HSP-70 mRNA
relative to GAPDH in the LAD region of hibernating animals versus
sham-instrumented controls. HSP-70 levels were uniform in all
transmural layers of the normally perfused regions as well as in the
LAD region in shams. In contrast, there was a greater than twofold
induction of HSP-70 mRNA in the subendocardium of hibernating hearts,
which decreased toward the subepicardium
(P < 0.05 vs. each corresponding layer in the normal region). Figure 7
summarizes the results of subendocardial HSP-70 mRNA in comparison to
mRNA levels of the cytosolic proteins calmodulin and
-myosin heavy
chain. The levels of HSP-70 mRNA were increased in hibernating compared
with sham animals. There was no regional induction of calmodulin and
-myosin heavy chain mRNA in the LAD region of hibernating animals
relative to normal remote regions. Whereas sham-instrumented animals
showed no regional difference in mRNA for HSP-70 or calmodulin, there was a slight but significant increase in the expression of
-myosin heavy chain that was secondary to increased levels in the LAD zones of
sham-instrumented animals.

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Fig. 5.
Expression of heat shock protein (HSP)-70 mRNA in representative
Northern blot from hibernating animal. Total RNA from subendocardium
(Endo), mid-myocardium, and subepicardium (Epi) of normal and
hibernating regions demonstrated uniform intensity of the GAPDH signals
in all layers confirming equal RNA loading. There was weak and uniform
hybridization for HSP-70 in normally perfused regions that did not vary
across the wall of the heart. In contrast, HSP-70 was induced in the
hibernating LAD regions. There was a distinct transmural pattern with
induction of HSP-70 mRNA most pronounced in subendocardial layers and
least marked in subepicardial layers.
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Fig. 6.
Summary of induction of HSP-70 mRNA in hibernating
(A,
n = 20) vs.
sham-instrumented (B,
n = 11) animals. Hibernating animals
had a more than twofold induction of HSP-70 mRNA in subendocardium.
This progressively decreased in outer layers of the myocardial wall but
remained significantly higher than corresponding normal remote region.
In contrast, HSP-70 expression in LAD and normal regions from
sham-instrumented animals was uniform across myocardial wall.
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Fig. 7.
Comparison of subendocardial HSP-70 induction to the cytosolic proteins
-myosin heavy chain and calmodulin in hibernating
(n = 20) vs. sham-instrumented
(n = 10) animals. There was twofold
relative induction (LAD/normal) of HSP-70 in hibernating animals,
whereas mRNA levels of calmodulin and -myosin heavy chain remained
unchanged. There was no alteration in HSP-70 or calmodulin mRNA in sham
animals, but a small increase in -myosin heavy chain expression was
statistically significant.
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|
Alterations of candidate proteins in hibernating myocardium.
Figure 8 and Table 3 summarize the
Western blot analysis for SR
Ca2+-ATPase, calsequestrin,
phospholamban, and HSP-70 protein from four hibernating animals.
Subgroup analysis showed no differences in resting or vasodilated flow
compared with the group used for mRNA measurements. In comparison to
normally perfused remote myocardium, hibernating regions had
significantly lower protein levels of SR
Ca2+-ATPase and phospholamban but
no significant change in calsequestrin. Immunoblots for HSP-70 in
hibernating regions revealed two bands consistent with the constitutive
(73 kDa) and inducible (72 kDa) forms. Although densitometric analysis
revealed significant increases in both forms, induction was most
evident in the 72-kDa band with a twofold increase. Thus the
quantitation of candidate proteins confirmed directionally similar
changes as seen for the mRNA.

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Fig. 8.
Western blot analysis of SR
Ca2+-ATPase, calsequestrin,
phospholamban, and HSP-70 in hibernating (H) vs. normal remote regions
(Nl). All 4 animals demonstrated reduction in SR
Ca2+-ATPase protein (105-110
kDa) and phospholamban (monomeric form 6 kDa), whereas calsequestrin
remained unchanged. HSP immunoblots demonstrated both 72- and 73-kDa
bands that were increased in hibernating LAD regions in comparison to
normally perfused remote regions.
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|
Relation of SR gene expression to regional adenosine flow reserve.
Figure 9 highlights the relation between relative
adenosine flow (LAD/normal) and mRNA changes for selected SR proteins.
A novel observation was that the magnitude of mRNA changes in
dysfunctional LAD regions correlated with the physiological
significance of the stenosis or collateral vasodilator reserve.
Sham-instrumented animals showed no relation between mRNA and relative
flow during adenosine. In contrast, the expression of phospholamban and
the SR Ca2+-ATPase mRNA were
significantly correlated with the severity of flow reserve reduction.
At modest levels of flow impairment, there appeared to be little
systematic change in mRNA despite the fact that myocardial function was
chronically depressed. When flow reserve was more severely reduced,
there was a reduction in SR Ca2+-ATPase and phospholamban
mRNA. Calsequestrin expression was unchanged over a wide range of
stenosis severity. These results suggest that the molecular adaptations
seen in hibernating myocardium are likely related to repetitive
ischemia that will increase in frequency and severity as
adenosine flow reserve is reduced.

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Fig. 9.
Relation between SR protein mRNA levels and relative subendocardial
flow during adenosine. A:
phospholamban. B: SR
Ca2+-ATPase.
C: calsequestrin. Variability in mRNA
expression for phospholamban and SR
Ca2+-ATPase was to a large extent
related to variations in physiological significance of flow limitation
to hibernating regions. Animals in which adenosine vasodilation was
most severely limited had the largest reductions in the expression of
SR uptake proteins. There was a poorer correlation between mRNA levels
and resting subendocardial flow (data not shown) [relative
phospholamban expression = 0.39 ln(relative resting flow) + 0.93, r = 0.45, P < 0.01; relative SR
Ca2+-ATPase expression = 0.26 ln(relative resting flow) + 0.96, r = 0.39, P < 0.01; relative
calsequestrin mRNA = 0.051 ln(relative resting flow) + 1.06, r = 0.09, P = ns].
|
|
 |
DISCUSSION |
There are two important new findings from our study. First, pigs with
hibernating myocardium develop regional alterations in the expression
of the SR calcium uptake proteins phospholamban and
Ca2+-ATPase, which may be one of
the mechanisms responsible for producing resting regional dysfunction.
Western analysis confirmed that the regional alterations in mRNA were
associated with regional changes in protein content. Whereas the
pattern of expression we found was similar to that reported in
cardiomyopathy and decompensated left ventricular hypertrophy, the
changes were regional and occurred without global alterations in
hemodynamics compared with sham control animals, indicating that they
arise from chronic intermittent myocardial ischemia. Second, we
found a regional induction of stress protein HSP-70 with increases in
both the 72- and 73-kDa proteins by Western blot analysis. To our
knowledge, this is the first demonstration that chronic reversible
subendocardial ischemia can alter myocyte phenotype on a
regional basis.
Downregulation in expression of selected SR proteins in hibernating
myocardium.
The results of our study indicate that the SR proteins involved in the
uptake and release of calcium, the SR
Ca2+-ATPase, phospholamban, and
the ryanodine receptor are coordinately downregulated in our porcine
model of hibernating myocardium. The changes were restricted to the
dysfunctional LAD region and did not appear to be related to a
generalized reduction in the SR proteins or a loss of SR within
myocytes because mRNA and protein levels for the SR calcium binding
protein calsequestrin remained unchanged. They also appear unrelated to
myocyte loss because mRNA levels for cytosolic proteins such as GAPDH,
calmodulin, and
-myosin heavy chain were not significantly reduced
in hibernating myocardium. Histological samples showed no myocardial
necrosis, and point counting showed only a small increase in connective tissue staining in the hibernating regions, similar to that reported in
patients with collateral-dependent myocardium distal to chronic LAD
occlusions and clinically documented hibernating myocardium (5). These
changes were much less extensive than those reported by Elsässer
et al. (10) where up to 58% of the wall was fibrotic in reversibly
dyssynergic myocardium, indicating that our findings may be
representative of an earlier stage of disease. Thus the selective
reduction in the expression of SR calcium uptake and release proteins
supports the hypothesis that the chronic contractile abnormalities
found in hibernating myocardium are secondary to SR dysfunction and
arise from chronic intermittent ischemia. This precedes the
advanced structural changes that could affect contractile function in
an independent fashion.
Previous experimental studies have reported directionally different
effects of acute ischemia on mRNA levels for SR proteins in
stunned myocardium. Frass et al. (14) examined the effects of two
10-min LAD occlusions in pigs. There was a regional induction of mRNAs
for the SR Ca2+-ATPase,
phospholamban, and calsequestrin that appeared to be transient in
nature with relative mRNA levels increasing to a maximum of about 1.5 to 2 times normal. Transcriptional assays also demonstrated induction
of mRNAs for a number of other proteins, including GAPDH, calmodulin,
and
-myosin heavy chain, suggesting an even more generalized pattern
of injury-repair following repetitive total occlusions (19). In
contrast, Abraham et al. (1) recently found no change in mRNA levels
for SR calcium regulatory proteins in stunned myocardium following
partial coronary occlusion, indicating that the effects of acute
ischemia on transcription may depend on the severity of flow
reduction. Other laboratories have demonstrated that phospholamban and
the SR Ca2+-ATPase protein levels
are unaltered following acute ischemia (26, 42). Thus the
findings in acutely dysfunctional, stunned myocardium are in sharp
contrast with the pattern of downregulation that we found in chronic
hibernating myocardium, indicating that the contractile dysfunction is
probably not simply a reflection of acute stunning that has not had
time to fully recover.
Interestingly, the regional mRNA and protein changes we found in
hibernating myocardium are similar to the global reductions in
expression of the SR Ca2+-ATPase
and phospholamban found in explanted hearts from patients with
end-stage congestive heart failure (29, 30). The ryanodine receptor has
also been found to be downregulated in ischemic but not idiopathic
dilated cardiomyopathy (7). Variability has been found regarding
corresponding changes in protein levels in advanced heart failure with
some studies showing them to be reduced (30), whereas others show them
to be unchanged (32, 39); these have recently been reviewed (16).
We found significant reductions in mRNA and protein levels for the SR
Ca2+-ATPase and phospholamban with
no change in calsequestrin in hibernating myocardium. These were
inversely related to coronary flow reserve and thus the propensity of a
region to develop ischemia. They did not appear to be the
result of reduced resting flow since we have recently reported regional
reductions in SR Ca2+-ATPase and
phospholamban in chronically stunned pigs that were studied 1 and 2 mo
after placement of an LAD stenosis (23). At this time animals had
depressed function and reduced LAD flow reserve with normal resting
perfusion (11). Thus the reduction in selected SR protein expressions
support the induction of a chronic adaptive response that develops in
relation to the physiological severity of a coronary stenosis.
Although acute inotropic stimulation improves myocardial function in
hibernating myocardium, the net effect is a complex interplay between
the cellular mechanisms responsible for chronic contractile dysfunction
and superimposed acute demand-induced ischemia. The relative
importance of altered myocyte function versus limited flow reserve in
the inotropic response will require additional studies where regional
metabolism can be quantified simultaneously. The regional reductions in
SR proteins found in hibernating myocardium where coronary flow reserve
is critically impaired may serve as an adaptive role because they could
lead to a regional attenuation of rate-related increases in myocardial
oxygen consumption during tachycardia vis-a-vis an altered force
frequency relation (17, 34). This could minimize regional imbalances
between myocardial oxygen supply and demand in a hibernating region
during stress. Although speculative, our data in hibernating myocardium
without heart failure raise the possibility that SR dysfunction may be a common and early abnormality in the evolution of ischemic
cardiomyopathy that is potentially reversible before the onset of
structural fibrosis.
Transmural induction of HSP-70 distal to chronic stenosis.
We also found that the stress protein HSP-70 was variably increased in
hibernating myocardium. The magnitude of HSP-70 mRNA induction was most
pronounced in the subendocardial layers. Previous studies have
demonstrated that HSP-70 can be induced after repetitive brief total
coronary occlusions in pigs and that this is associated with
accelerated recovery of myocardial function when a series of brief
occlusions is performed 24 h later (preconditioning against myocardial
stunning) (43). Other studies have demonstrated a small, statistically
insignificant reduction in infarct size in pigs following prolonged
occlusions when a repetitive ischemic stimulus is performed 24 h
earlier (second window of preconditioning)(35). Despite these
experimental findings, the relevance of this endogenous protective
mechanism in chronic coronary artery disease is uncertain since
ischemia produced by brief repetitive total coronary occlusions is somewhat artificial and, aside from unstable coronary syndromes, unlikely to mimic clinical circumstances in which the myocardium will
be subjected to demand-induced ischemia.
Our study extends these earlier observations by demonstrating that
HSP-70 can be chronically induced distal to a severe coronary stenosis
or in collateral-dependent myocardium. Nevertheless, induction of
HSP-70 required a severe reduction in coronary flow reserve. Western
blot analysis revealed a small (10%) but significant increase in the
constitutive 73-kDa form and a twofold increase in the inducible 72-kDa
isoform. Variability in HSP-70 expression appears importantly related
to the physiological significance of the chronic coronary stenosis in
this model of hibernating myocardium. In the subendocardium, where
coronary flow during adenosine was critically reduced, HSP-70 mRNA was
more than two times greater than normal regions. Even though maximal
flow was also severely restricted in subepicardial hibernating LAD
regions, the level of HSP-70 induction was considerably lower (1.3 times normal). Thus the chronic induction of HSP-70 appears to require a physiologically severe reduction in coronary flow reserve and may not
be spontaneously induced in the majority of patients with moderate
chronic coronary disease in which resting contractile function is normal.
Methodological limitations.
We assessed regional wall motion with left ventriculography to
circumvent potential nonspecific effects of instrumentation on inducing
mRNA (6). Because the relation between regional perfusion and radial
wall motion has not been established, we could not ascertain the extent
to which chronic recurrent myocardial stunning (which would be
associated with a dissociation between subendocardial flow and the
degree of dysfunction) could have contributed to the chronic
dysfunction. Nevertheless, stunning alone cannot explain our
observations since it is associated with normal rather than reduced
values of resting perfusion (4, 8, 36, 41). In pigs studied at 1 and 2 mo after instrumentation we have found viable dysfunctional myocardium
without a reduction in resting flow (11). These data along with the
present findings are consonant with the notion that there is a temporal
transition from chronic stunning with normal flow to hibernation with
depressed resting flow during the progression of a chronic stenosis, as we previously reported in dogs (8).
The presence of recruitable inotropic reserve in response to
catecholamine infusion, the regional increase in
18F-2-deoxyglucose uptake with a
mismatch pattern similar to humans (28) in our previous study (12), and
the lack of pathological necrosis all substantiate that the
dysfunctional LAD regions were viable. The molecular alterations in
hibernating myocardium would support the notion that this involves
cellular remodeling and may take considerably longer than the hours to
days over which stunning improves. Further studies will be required to
examine the time course of improvement following the restoration of
blood flow in this model.
In summary, although the physiological implications of reduced
expression of selected SR proteins and increased expression of HSP-70
in hibernating myocardium remain to be established, the intriguing
possibility exists that myocytes subjected to chronic reversible
ischemia may be protected against the development of irreversible injury. Adaptations in SR gene expression could be responsible for the chronic alterations in regional mechanical function
and reduce regional myocardial demand. In conjunction with known
variables such as collateral flow and myocardial oxygen consumption at
the time of ischemia, the induction of molecular adaptations
that increase the tolerance of the heart to inadequate perfusion in
chronic ischemic heart disease may be responsible for the beneficial
effects of reperfusion as late as 12 h after a coronary occlusion.
Further studies will be required to determine the physiological
importance of these and other intrinsic myocardial adaptations in
hibernating myocardium.
 |
ACKNOWLEDGEMENTS |
We thank Deana Gretka, Amy Johnson, Felicia Bosinski, and Susan
Fopeano for expert technical assistance throughout this study.
 |
FOOTNOTES |
This work was supported by a Clinician Scientist Award and Affiliate
Grant-in-Aid from the American Heart Association; a Merit Review Award
from the Office of Research and Development, Medical Research Service,
Dept. of Veterans Affairs; the Albert and Elizabeth Rekate Fund; and
the National Heart, Lung, and Blood Institute Grant HL-55324. S. Jacob
was supported by the John C. Sable Award from the New York State
Independent Order of Odd Fellows.
Portions of this work were presented in part at the 1995 American
Federation for Clinical Research Meeting (San Diego, CA), the 1995 Scientific Sessions of the American Heart Association (Anaheim, CA),
and the 1997 Scientific Sessions of the American College of Cardiology
(Anaheim, CA).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. M. Canty,
Jr., Biomedical Research Bldg., Rm. 347, Div. of Cardiology, Dept. of
Medicine, Univ. at Buffalo, 3435 Main St., Buffalo, NY 14214 (E-mail:
canty{at}buffalo.edu).
Received 18 February 1999; accepted in final form 24 May 1999.
 |
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