Persistent Regional Downregulation in Mitochondrial
Enzymes and Upregulation of Stress Proteins in Swine
With Chronic Hibernating Myocardium
Brian Page, Rebeccah Young, Vijay Iyer, Gen Suzuki, Maciej Lis, Lioubov Korotchkina,
Mulchand S. Patel, Kenneth M. Blumenthal, James A. Fallavollita, John M. Canty, Jr
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Abstract—Hibernating myocardium is accompanied by a downregulation in energy utilization that prevents the immediate
development of ischemia during stress at the expense of an attenuated level of regional contractile function. We used a
discovery based proteomic approach to identify novel regional molecular adaptations responsible for this phenomenon in
subendocardial samples from swine instrumented with a chronic LAD stenosis. After 3 months (n⫽8), hibernating
myocardium was present as reflected by reduced resting LAD flow (0.75⫾0.14 versus 1.19⫾0.14 mL 䡠 min⫺1 䡠 g⫺1 in remote)
and wall thickening (1.93⫾0.46 mm versus 5.46⫾0.41 mm in remote, P⬍0.05). Regionally altered proteins were quantified
with 2D Differential-in-Gel Electrophoresis (2D-DIGE) using normal myocardium as a reference with identification of
candidates using MALDI-TOF mass spectrometry. Hibernating myocardium developed a significant downregulation of many
mitochondrial proteins and an upregulation of stress proteins. Of particular note, the major entry points to oxidative
metabolism (eg, pyruvate dehydrogenase complex and Acyl-CoA dehydrogenase) and enzymes involved in electron transport
(eg, complexes I, III, and V) were reduced (P⬍0.05). Multiple subunits within an enzyme complex frequently showed a
concordant downregulation in abundance leading to an amplification of their cumulative effects on activity (eg, “total” LAD
PDC activity was 21.9⫾3.1 versus 42.8⫾1.9 mU, P⬍0.05). After 5-months (n⫽10), changes in mitochondrial and stress
proteins persisted whereas cytoskeletal proteins (eg, desmin and vimentin) normalized. These data indicate that the proteomic
phenotype of hibernating myocardium is dynamic and has similarities to global changes in energy substrate metabolism and
function in the advanced failing heart. These proteomic changes may limit oxidative injury and apoptosis and impact
functional recovery after revascularization. (Circ Res. 2008;102:103-112.)
Key Words: metabolism 䡲 proteomics 䡲 hibernating myocardium 䡲 ischemic heart disease
H
ibernating myocardium is characterized by viable,
chronically dysfunctional myocardium that develops in
response to repetitive myocardial ischemia.1,2 We have previously demonstrated that the relation between regional
oxygen consumption, coronary flow, and function in response
to stress is attenuated in hibernating myocardium and thus
dissociated from the usual determinants of myocardial oxygen demand.3 By reducing regional energy utilization, hibernation prevents the development of ischemia after submaximal stress. This is supported by a lack of biochemical markers
of ischemia and preservation of total ATP and creatine
phosphate content in swine with hibernating myocardium3,4
as well as human biopsies from patients without significant
fibrosis.5 Although there has been interest in identifying the
role of increased glucose uptake in these responses, maximal
insulin stimulated glucose uptake is unchanged in chronic
hibernating myocardium, and alterations in other metabolic
pathways responsible for the attenuated increase in oxygen
consumption are unknown.2,4,6,7
We hypothesized that hibernating myocytes in viable
dysfunctional myocardium can intrinsically downregulate
their metabolic needs to achieve a balance between supply
and demand at a reduced regional workload. As a first step to
delineate the metabolic pathways involved, we used a discovery based proteomic approach to identify targets that are
regionally modified in pigs with chronic hibernating myocardium. Enzymatic activity assays of the pyruvate dehydrogenase complex (PDC) were used to illustrate how changes in
the expression of multiple protein components of an enzyme
system can combine to produce a cumulative functional
impact in vitro. Lastly, because depressed flow and function
remain constant for at least 2 months in this model8 we
assessed the stability of protein changes after the development of hibernating myocardium. The results demonstrate a
chronic downregulation of mitochondrial enzymes that, while
regional and associated with normal global function, is
similar to that in the advanced failing heart.
Original received May 10, 2007; revision received October 3, 2007; accepted October 17, 2007.
From the VA WNY Health Care System, the Center for Research in Cardiovascular Medicine, the Center for Excellence in Bioinformatics and Life
Sciences, the Departments of Medicine, Physiology and Biophysics, and Biochemistry at the University at Buffalo, NY.
Correspondence to John M. Canty Jr, MD, Division of Cardiovascular Medicine, University at Buffalo, Biomedical Research Building, Room 361,
3435 Main St, Buffalo, NY 14214. E-mail [email protected]
© 2008 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.107.155895
103
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Circulation Research
January 4/18, 2008
MW
80 kD -
50 kD -
Figure 1. Sypro Ruby–stained 2D-gel used
for spot identification with MALDI-TOF.
Whole tissue protein preparations were
separated by isoelectric focusing (nominal
pI 3 to 10) and molecular weight (nominal
range 15 kDa to 100 kDa).
30 kD -
20 kD -
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4.5
6
9
pI
Materials and Methods
Procedures and protocols conformed to institutional guidelines for
the care and use of animals in research and are detailed in the online
supplement. Briefly, farm-bred juvenile pigs were chronically instrumented with a 1.5- to 2.0-mm fixed diameter stenosis placed on the
proximal left anterior descending (LAD) coronary artery. This model
progresses from chronically stunned to hibernating myocardium after
3 months with physiological features persisting unchanged for up to
5 months after instrumentation.2,8,9 Sham animals were normal or
underwent thoracotomy and dissection of the LAD without placement of an occlusive stenosis. Hibernating animals were studied 3
months (n⫽8) or 5 months (n⫽10) after instrumentation in the
closed-chest sedated state (Telazol/xylazine IM and propofol 2 to 5
mg 䡠 kg⫺1 䡠 min⫺1IV). Resting and vasodilated coronary flows were
assessed using fluorescent microspheres as previously described, and
LV function was assessed with M-mode echocardiography.10 To
circumvent acute posttranslational modifications arising from pharmacological stimulation,11 animals were recovered and reanesthetized 72 hours later with hearts excised in the unstimulated state.
Samples were flash frozen and total protein extracted from approximately 0.2g of myocardial tissue.
2D Differential-in-Gel Electrophoresis (2D-DIGE)
We used 2D-DIGE for analysis of changes in myocardial protein
expression (Figure 1).12 Reagents, electrophoresis materials, and
software were all obtained from GE Healthcare. Methodological
details are provided in the online supplement (available online at
http://circres.ahajournals.org).
Samples were labeled with CyDye DIGE Fluor (Cy3 or Cy5), and
an internal standard (Cy2 labeled) contained an equal mix of protein
from all animals or a pooled sham sample. Paired comparisons of
LAD versus remote (3 months n⫽8; sham n⫽7) as well as unpaired
LAD versus sham (3 months n⫽8; 5 months n⫽10) were performed
on separate gel runs. For all analyses, Cy3 and Cy5 were randomly
used to label LAD and control regions to prevent dye bias. Proteins
were isoelectrically focused with immobilized pH gradient strips (24
cm, pH 3 to 10, nonlinear) using an IPGphor system, and subsequently electrophoresed on 12.5% polyacrylamide gels in an Ettan
DALT SDS-PAGE system. Gels were scanned with a Typhoon 9410
imager, cropped (ImageQuant v5.2), and imported into DeCyder
DIA software (v5.0) for spot identification and normalization. Spot
analysis was performed using DeCyder BVA software.
MALDI-TOF and Protein Identification
Spots were excised from separate 2D-gels loaded with 500 to 1000 ␮g
of total protein and stained with Sypro Ruby (Molecular Probes). In-gel
digestion was performed using trypsin. Digests were concentrated and
purified with ZipTips (Millipore), and eluted in a saturated solution
of ␣-cyano-4-hydroxycinnamic acid. We used a Bruker Daltonics Biflex
MALDI-TOF mass spectrometer (Bruker Daltonics) or a Bruker
Daltonics Autoflex MALDI-TOF in TOF-TOF mode for peptide mass
fingerprinting. Proteins were identified using Mascot. MOWSE scores
greater than 61 were significant for the Swiss Protein database, whereas
scores of greater than 67 were significant for the NCBI database
(Pⱕ0.05). The reported scores and accession numbers are from the
Swiss Protein database unless otherwise specified. For MALDI-TOFTOF data, the MS Ion search feature was used with the MSDB database.
Scores of 39 or higher were significant.
Pyruvate Dehydrogenase Complex, Cytochrome
C Oxidase, and Citrate Synthase Activity
Methodological details are provided in the online supplement.
“Active” and “total” PDC activity were measured using 14CO2
collected during in vitro reaction with radioactivity measured with a
scintillation counter. Dihydrolipoamide dehydrogenase (E3), cytochrome c oxidase and citrate synthase activity were measured
spectrophotometrically.
Statistical Analysis
Data are presented as the mean⫾SEM. Student’s paired t test was
used to examine differences between LAD and remote regions of the
same heart. For serial changes in hibernating myocardium evaluated
3 and 5 months after instrumentation, we used unpaired t tests. A
probability value of ⬍0.05 was significant.
Results
In both 3- and 5-month swine with hibernating myocardium,
TTC staining showed no evidence of infarction. Assessment
of connective tissue demonstrated 4.7⫾0.6% in full-thickness
samples taken from the hibernating LAD region compared
with 3.0⫾0.3% for remote regions.
Flow and Function in 3- and 5-Month Swine With
Hibernating Myocardium
Hemodynamics are summarized in the online supplement and
were similar in each group. Transmural myocardial perfusion is
Page et al
3 Months
Flow (ml/min/g))
Rest
A
5 Months
LAD
Remote
2
1
*
*
* p<0.05 LAD vs. Remotee
*
*
0
Adenosine
Flow (ml/min/g)
6
4
*
2
*
*
*
*
*
*
*
0
Endo
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ESWT – EDWT (mm)
B
Mid
Epi
FT
3 Month Wall Thickening
g
8
Endo
Mid
Epi
FT
5 Month Wall Thickening
LAD
Remote
* p<0.05 LAD vs. Remote
6
4
*
*
2
0
Figure 2. Flow and function in swine with hibernating myocardium. A, Regional perfusion. At 3 months there was a significant
reduction in resting subendocardial flow in the hibernating region
versus remote normally perfused myocardium that persisted in animals studied at 5 months. Adenosine vasodilated flow was significantly reduced in the LAD region and unable to increase above
resting values in the subendocardium. There were no temporal
differences except for a small increase in remote resting flow after
5 months (P⬍0.05) vs 3 months. B, Both 3- and 5-month swine
demonstrated a significant reduction in LAD function relative to normal
remote regions. There were no significant differences between animals with hibernating myocardium studied at 3 and 5 months.
summarized in Figure 2A. Resting subendocardial flow was
reduced in both 3- and 5-month swine (LAD 0.75⫾0.14 versus
1.19⫾0.14 mL 䡠 min⫺1 䡠 g⫺1 in 3-month swine; LAD 0.84⫾0.07
versus 1.65⫾0.15 mL 䡠 min⫺1 䡠 g⫺1 in 5-month swine, both
P⬍0.05). After adenosine vasodilation subendocardial flow was
critically impaired and failed to increase above resting levels.
Global function was normal and there was no clinical evidence
of heart failure in either group. Regional function is summarized
in Figure 2B. LAD wall thickening in hibernating LAD regions
was reduced relative to remote myocardium in both groups
(3-month LAD 1.93⫾0.46 mm versus 5.46⫾0.4 mm and
5-month LAD 2.73⫾0.35 versus 6.93⫾0.57; P⬍0.05 for both).
With the exception of a small increase in resting LAD flow
(P⬍0.05), hemodynamics, function, and perfusion were similar
in swine studied at 3 or 5 months after instrumentation.
Regional Proteomic Changes in Hibernating
Myocardium at 3-Months Paired Analysis
With Remote Myocardium
To identify differentially expressed proteins, we initially used
a paired analysis in the 3-month hibernating group using the
Proteomic Analysis of Hibernating Myocardium
105
remote normally perfused region from each heart as an
internal control for potential posttranslational modifications
related to tissue harvesting. Detailed quantitative analyses
and protein identification data are provided in the online
supplement. Whole tissue protein preparations revealed 1323
unique protein spots with 1019 detectable in at least half of
the 2D gels. Of these, 191 were significantly altered
(P⬍0.05) in hibernating LAD regions (70 increased and 121
decreased). We identified 52 differentially expressed protein
spots representing 37 unique proteins (supplemental Table I).
An additional 62 spots have been identified which were not
significantly altered representing 35 unique control proteins.
For all identified spots, the average Mascot Mowse score for
each protein was 99 with 25% average peptide sequence
coverage. Of the 72 unique proteins identified, 35 had known
porcine sequences.
Abundance of hibernating LAD proteins relative to remote
regions in hibernating (n⫽8) and sham animals (n⫽7) is
tabulated (supplemental Table II) with selected examples
summarized in Figure 3A and 3B. We observed a general
downregulation of mitochondrial proteins including citric
acid cycle enzymes (6 unique proteins) and electron transport
chain and ATP-synthase subunits (8 unique proteins). There
was also a downregulation of cytoplasmic CK, myoglobin,
and long chain Acyl-CoA dehydrogenase. Glycolytic proteins
were not altered with the exception of pyruvate kinase which
was reduced. Contractile proteins such as myosin heavy
chain, myosin light chain isoforms, troponin T, and tropomyosin exhibited multiple spots on the gels likely reflecting
posttranslational modifications of which most were unchanged or reduced in hibernating myocardium. Regional
differences in protein expression between LAD and remote
regions of sham animals were infrequent and small (supplemental Table II).
In contrast to the regional reductions in mitochondrial
proteins in hibernating myocardium, there were significant
increases in stress proteins including ␣B-crystallin, HSP27,
and HSP20-␤6. Likewise, cytoskeletal proteins including
desmin and vimentin, and antioxidant proteins such as superoxide dismutase and peroxiredoxin-2, were also increased
early after the development of hibernating myocardium.
Amplification of Protein Subunit Changes for PDC
Many of the proteins identified were subunits of a given
protein complex of which we chose PDC as an example to
illustrate the cumulative impact on biological activity (Figure
4). We found modest reductions in individual PDC components by 2D-DIGE but their effects on PDC activity were
more pronounced. “Total” PDC activity (LAD 21.9⫾3.1
versus 42.8⫾1.9 nmoles/mg/min, P⬍0.05) and E3 catalytic
activity (1.04⫾0.11 versus 1.47⫾0.30 ␮moles/mg/min,
P⬍0.05) were significantly downregulated in samples taken
from hibernating LAD regions. The greater reductions in
PDC activity in hibernating myocardium reflected the net
result of changes in the individual subunits as well as other
unidentified regulatory components.
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A
January 4/18, 2008
Mitochondrial Proteins
Pyruvate
Dehydrogenase
E1 β subunit
1.1
Long Chain
Acyl-CoA
Dehydrogenase
Sham, n=7
3 Months, n=8
* p<0.05 vs. Remote
*
*
LAD 0.9
Remote 0.7
NAD+
Isocitrate
Dehydrogenase
*
Malate
Dehydrogenase
(mitochondrial)
*
0.5
Cytochrome Bc 1
– Complex III
NADH
Dehydrogenase
ATP Synthase
chain
α-chain
ATP Synthase
-chain
β-chain
1.1
*
*
LAD 0.9
Remote
*
*
0.7
B
Cytosolic/Structural Proteins
GAPDH
Cytoplasmic
CK M
Myoglobin
Pyruvate Kinase
1.1
*
*
Annexin 2
Vimentin
LAD 0.9
Remote
*
0.7
0.5
alpha B Crystallin
2.5
*
*
*
Desmin
Sham, n=7
3 Months, n=8
* p<0.05 vs. Remote
*
LAD
1.5
Remote
0.5
Temporal Evolution of Proteomic Changes in
Chronic Hibernating Myocardium-Unpaired
Analysis With Normal Controls
Pyruvate Dehydrogenase Complex Enzyme Activityy
15
10
5
0
Remote
LAD
50
nmole/mg protein/min
n
20
‘Total’ PDC
Activity
*p<0.05 vs. Remote
40
30
20
10
0
Dihydrolipoamide
Dehydrogenase
(E3) Activity
*
µmole/mg protein/min
n
‘Active’
PDC
Activity
% ‘Total’ Activity
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0.5
Figure 3. Differential protein expression in
swine after the development of hibernating
myocardium (3-month paired LAD versus
remote analysis). A, Mitochondrial protein
expression represented as LAD/Remote ratios
from 2D-DIGE for swine with hibernating myocardium (n⫽8) versus normal sham animals
(n⫽7). Entry points to oxidative metabolism as
well as citric acid cycle and electron transport
chain enzymes were regionally reduced in
hibernating myocardium. B, Cytosolic/structural
protein expression. GAPDH was not altered but
cytoplasmic CK-M and myoglobin were regionally reduced in hibernating myocardium. In
contrast, there was upregulation of stress and
cytoskeletal proteins. Additional proteins are
summarized in the online supplement.
1.6
1.2
*
0.8
0.4
0
Figure 4. Pyruvate dehydrogenase complex (PDC) and E3 activity. “Total” PDC was more markedly reduced than the corresponding relative reduction in protein abundance by 2D-DIGE.
Likewise, reductions in E3 catalytic activity were significant
whereas E3 protein levels by 2D-DIGE were not.
To determine the chronicity of the proteomic changes in
hibernating myocardium we assessed protein expression ratios in LAD versus normal sham animals studied after 3 and
5 months. Data are summarized in supplemental Table II with
selected proteins in Figure 5A and 5B. Of the 114 spots
identified, 25 showed significant differences at 5 months in
comparison with swine studied early after the development of
hibernating myocardium (3 months). The trends were predominantly related to the initial differential protein expression normalizing. We found persistent reductions in mitochondrial protein expression in 5-month animals compared
with 3-month animals with notable exceptions including long
chain acyl-CoA dehydrogenase, ATP synthase, mitochondrial
CK, and mitochondrial aspartate aminotransferase, all of
which returned toward normal at 5 months. Pyruvate kinase
became significantly downregulated at 5 months. Whereas
increases in cytoskeletal proteins at 3 months normalized
Page et al
A
Proteomic Analysis of Hibernating Myocardium
107
Mitochondrial Proteins * p<0.05 vs. Normal
Sham, n=7
3 Months, n=8
5 Months, n=10
† p<0.05 3 month vs. 5 month
Pyruvate
Dehydrogenase
E1 β subunit
Long Chain
Acyl-CoA
Dehydrogenase
1.1
NAD+
Isocitrate
Dehydrogenase
Malate
Dehydrogenase
(mitochondrial)
†
0.9
LAD 0.7
Normal
*
* *
*
0.5
0.3
NADH
Dehydrogenase
Cytochrome
Bc1 Complex III
ATP Synthase
α-chain
-chain
ATP Synthase
β-chain
chain
1.1
0.9
*
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LAD 0.7
Normal
*†
*
0.5
0.3
B
Sham, n=7
3 Months, n=8
5 Months, n=10
Cytosolic/Structural Proteins * p<0.05 vs. Normal
† p<0.05 3 month vs. 5 month
GAPDH
Cytoplasmic
CK-M
Myoglobin
Pyruvate Kinase
1.1
Figure 5. Temporal changes in LAD protein expression in animals with persistent
hibernating myocardium studied 5 months
after instrumentation (Hibernating LAD
versus Sham LAD). Sham animals referenced to normal remote regions are the
same as Figure 3. A, Reductions in most
mitochondrial proteins were similar at 3
months (n⫽8) and 5 months (n⫽10). In
contrast, long chain Acyl-CoA dehydrogenase and ATP synthase-␤-chain tended to
normalize. B, Cytosolic proteins also
remained depressed at both time points,
whereas GAPDH was unchanged in all
groups. Although stress proteins remained
persistently increased, the increases in
cytoskeletal proteins normalized after 5
months. Additional proteins are summarized in the online supplement.
0.9
LAD
0.7
Normal
*†
*
0.5
*
0.3
Alpha β Crystallin
Annexin 2
Vimentin
Desmin
2.5
LAD 1.5
Normal
*
*
†
†
0.5
after 5 months, stress proteins such as ␣B-crystallin, HSP20␤6, and HSP-27 remained chronically elevated in hibernating
myocardium. Figure 6 summarizes the regional and temporal
alterations of mitochondrial proteins, and Figure 7 summarizes the results for cytoskeletal proteins early and late after
the development of hibernating myocardium.
Cytochrome C Oxidase, Citrate Synthase,
and Mitochondrial Protein Content in
Hibernating Myocardium
To further support the functional importance of protein
changes in hibernating myocardium identified from 2D-gels,
we performed activity assays for cytochrome c oxidase and
citrate synthase (Figure 8A). There was a reduction in activity
for both enzymes in the LAD region compared with sham
controls (Pⱕ0.05). Subendocardial mitochondrial and total
protein yield per gram of tissue was the same for all samples,
suggesting that reductions in mitochondrial mass/volume
were not the cause of these changes (Figure 8B).
Discussion
The present study used a discovery based proteomic approach
to demonstrate intrinsic downregulation of many of the
mitochondrial enzymes responsible for oxidative metabolism
and electron transport in response to a flow limiting coronary
stenosis. These changes may contribute to the reductions in
resting flow, function, and oxygen consumption that occur in
the absence of ischemia or infarction in chronic hibernating
myocardium. The chronic upregulation of stress proteins coupled with the transient increases in cytoskeletal proteins support
108
Circulation Research
January 4/18, 2008
Complex I
NADH
dehydrogenase
75 kDa subunit
Complex II
Complex II
Flavoprotein
subunit
Complex III
Cytochrome Bc1
core protein 1
Complex V
ATP Synthase
F1 α chain
Oxidative
Phosphorylation
Inner
mitochondrial
membrane
NADH
dehydrogenase
51 kDa subunit
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Mitochondrial
Matrix
Outer
mitochondrial
membrane
NADH
dehydrogenase
24 kDa subunit
ATP Synthase
F1 ß chain
Mitofilin
Protein
Transport/Assembly
Mitochondrial
Proteins
NADH
dehydrogenase
30 kDa subunit
Prohibitin
Pyruvate
dehydrogenase
Pyruvate
dehydrogenase
E1 α subunit
Pyruvate
dehydrogenase
E1 ß subunit
Pyruvate
dehydrogenase
E2
Dihydrolipoamide
dehydrogenase
E3
Citric Acid
Cycle
Aconitase
Isocitrate
dehydrogenase
α subunit
Malate
dehydrogenase
Dihydrolipoamide
succinyltransferase
Beta
Oxidation
Long chain
acyl CoA
dehydrogenase
Medium chain
acyl CoA
dehydrogenase
Metabolite
Catabolism
Isovaleryl CoA
dehydrogenase
Aspartate
aminotransferase
High Energy
Phosphate Metabolism
Voltage
dependent anion
channel 1
Voltage
dependent anion
channel 2
Protein
Synthesis
Cell
Proliferation
and aging
Succinyl CoA 3
ketoacid CoA
transferase 1
Mitochondrial
Creatine kinase
Translation
elongation factor
EF-Tu
NADP+ Isocitrate
dehydrogenase
47 kDa subunit
Mitochondrial
stress protein
70 (GRP 75)
Figure 6. Regional alterations in mitochondrial protein expression categorized by function, direction, and temporal change. The open
boxes indicate proteins that remain unchanged in hibernating myocardium at both time points. Solid blue depicts proteins that are significantly decreased in hibernating myocardium and solid yellow are increased. The hatched boxes indicate proteins that are significantly changed in hibernating swine studied at 5 months versus 3 months with most of the temporal changes being a diminution of differences present at 3 months versus normal. With rare exception, mitochondrial proteins were either unchanged or reduced.
the notion that the molecular mechanisms operative in hibernating myocardium are dynamic, protect myocytes from irreversible injury, and do not always lead to inexorable fibrosis.
Chronic Downregulation of Mitochondrial
Function and Oxygen Consumption in
Hibernating Myocardium
We found regional reductions in many of the enzymes
involved in oxidative metabolism in hibernating myocardium.
Interestingly, these changes involved entry points of carbohydrate (PDC) and fatty acid metabolism (acyl-CoA dehydrogenase) as well as many of the components of the electron
transport chain. The majority of these changes persisted in
swine where hibernating myocardium was present for at least
2 months (5-month animals). Changes in mitochondrial
proteins paralleled chronic reductions in flow and function in
the absence of progressive fibrosis as we have previously
demonstrated in this model over the same time frame.8 The
regional reductions in metabolic enzymes do not appear to be
related to myocardial scar as there was no significant infarction by TTC and only trivial regional increases in myocardial
connective tissue are found in this model. Furthermore,
although not all protein spots could be identified using mass
spectrometry, the majority of LAD protein spots were not
significantly altered when compared with normal myocardium. Unchanged metabolic enzymes that were identified
included GAPDH, triosephosphate isomerase, phosphoglycerate mutase, aminoacylase-1, and DRP-2. Thus, the regional
reductions in mitochondrial enzyme expression appear to
represent an intrinsic adaptation of the heart to hibernation.
There are in vivo as well as in vitro physiological correlates
of the reduced mitochondrial enzyme levels. In previous work
we have demonstrated that there is a reduction in myocardial
oxygen consumption at rest and during submaximal stress
that allows increases in external workload to occur without
immediately precipitating subendocardial ischemia in hibernating myocardium.3 This “protection” against the development of an oxygen supply/demand mismatch is, however,
accomplished at the expense of reduced regional LV function.
Using isolated mitochondria from a similar model, McFalls
demonstrated a reduction in the respiratory control index in
hibernating myocardium which they postulated to reflect a
basal uncoupling of mitochondrial respiration via increases in
uncoupling protein-2.13 They also demonstrated that mito-
Page et al
Glycolytic
Enzymes
Proteomic Analysis of Hibernating Myocardium
α – enolase
GAPDH
ß – enolase
Phosphoglycerate
mutase 2 M
isozyme
Fructose
bisphosphate
aldolase A
109
Muscle specific
phosphopyruvate
hydratase
Triosephosphate
isomerase
Structural
Proteins
Vimentin
Heat Shock
Related
Proteins
HSP 70
(70 kDa
Protein 1)
Superoxide
Dismutase
Antioxidant
Proteins
Cytosolic
Proteins
Oxygen carrying
proteins
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Anaerobic
Metabolism
MalateAspartate
Shuttle
Vinculin
Desmin
GRP 78
HSP 27
Actin
Interacting
Protein
HSP 20
beta-6
Pyruvate
Kinase
α B crystallin
HSP 60
Peroxiredoxin 2
High Energy
Phosphate
Metabolism
Myoglobin
Cytoplasmic
Creatine
Kinase
M chain
L lactate
dehydrogenase
B chain
Malate
dehydrogenase
Aspartate
Aminotransferase
Chaperones
Dehydropyrimidinases
Aminoacylases
RNA Binding
T-Complex
protein 1
(Chaperonin)
Dihydrpryimidinase
Related protein 2
(DRP-2)
Aminoacylase-1
Aconitase 1
(Fe regulatory
protein 1)
Figure 7. Regional alterations in cytosolic protein expression categorized by function, direction, and temporal change. The open boxes
indicate proteins that remain unchanged in hibernating myocardium at both time points. Solid blue are proteins that are significantly
decreased in hibernating myocardium and solid yellow are increased. Hatched boxes indicate proteins that are significantly altered in
swine studied at 5 months versus 3 months with most of the temporal changes being a diminution of regional differences present at 3
months to normal. Stress proteins and antioxidant proteins were increased in hibernating myocardium. Metabolic proteins involved in
glycolysis, creatine kinase, and myoglobin were reduced.
chondria from hibernating myocardium are better adapted to
ischemia as evidenced by decreased superoxide formation
and a preservation of state-3 mitochondrial oxygen consumption after a 20-minute period of anoxia-reoxygenation. Collectively, these observations support the central role of
mitochondrial respiration in the adaptation of the heart to
chronic repetitive ischemia.
The results of the present study may help to understand the
temporal pattern of myocyte apoptosis in chronic hibernating
myocardium. The progression from chronically stunned to
hibernating myocardium is associated with an increase in
myocyte apoptosis that leads to substantial regional myocyte
loss and compensatory cellular hypertrophy in animals with
hibernating myocardium that are studied at 3 months.14,15
Although the physiological findings of hibernating myocardium remain unchanged up to 5 months, data from our
previous studies demonstrated that apoptosis returns to levels
that are no different than normal myocardium (19⫾8 apoptotic myocytes/106myocytes versus 160⫾50 apoptotic myocytes/106 myocytes at 3 months, P⬍0.05).10,14 The reduction
in the frequency of apoptosis to levels similar to normal
animals supports the hypotheses that the downregulation in
mitochondrial oxidative enzymes and upregulation of stress
proteins serve to adapt the heart and prevent progressive
myocyte loss.
Comparison With Stunned Myocardium and
Pharmacological Preconditioning
The proteomic changes we found in chronic hibernating
myocardium differ substantially from those found in infarcted
hearts but have similarities as well as important differences
with findings that have been reported in isolated hearts with
acutely stunned myocardium and pharmacological preconditioning in isolated cardiac myocytes.11,16 White examined
isolated rabbit hearts subjected to 15 minutes of ischemia and
60 minutes of reperfusion using a proteomic approach.16
Similar to hibernating myocardium, spots representing stress
proteins such as HSP27 and ␣B-crystallin were increased
after acute ischemia. In contrast to the concordant downregulation of mitochondrial proteins identified in hibernating
myocardium, components of PDC and ATP synthase increased, whereas components of other enzymes such as
110
A
Circulation Research
January 4/18, 2008
Total
Cytochrome C Oxidase Activity
Total
Citrate Synthase Activity
Sham, n=10
LAD, n=9
Units/mg Protein
1.2
Remote, n=9
* p<0.05 vs. remote
0.8
† p<0.05 vs. sham
0.6
0.8
*†
†
0.4
0.4
0.2
0
B
0
Mitochondrial Protein Yield
Total Protein Yield
5
mg Protein/g Tissue
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Sham, n=10
LAD, n=9
Remote, n=9
Figure 8. A, Cytochrome C oxidase and citrate
synthase activities in 5-month animals with
hibernating myocardium and 5-month shams.
The activity of both enzymes was reduced in
the LAD region. B, Total mitochondrial protein
content per gram of tissue was similar in LAD,
remote, and normal myocardium as was total
protein content.
50
4
40
3
30
2
20
1
10
0
0
electron transport complexes I and II and VDAC-1 showed
bidirectional changes after acute ischemia. In isolated myocytes subjected to pharmacological preconditioning, Arrell
found selected components of proteins involved in oxidative
phosphorylation to increase.14 These included electron transport complex I, ATP synthase, isocitrate dehydrogenase, and
the pyruvate dehydrogenase E3-binding protein, whereas
other mitochondrial energetic proteins were unchanged. The
protein changes observed after acute interventions were
frequently in opposite directions from those in hibernating
myocardium and most likely reflected posttranslational modifications because the time period from intervention to tissue
harvesting was only 60 minutes in these studies. Nevertheless, the studies following acute ischemia and pharmacogical
preconditioning support a fundamentally different response
of the chronically adapted hibernating heart. Additional
studies in swine subjected to reversible ischemia will be
required to fully elucidate all of the differences and similarities in myocardial proteomics with hibernating myocardium
but should provide insight into whether stunning or preconditioning are components of the chronic adaptive response to
repetitive ischemia.
Regional Proteomic Changes in Hibernating
Myocardium Versus Global Changes in
Hypertrophied and Failing Myocardium
Interestingly, although this single vessel animal model of
hibernating myocardium is associated with normal global LV
function, it results in regional proteomic changes that are very
similar to the global changes that have been reported in
advanced congestive heart failure. Like hibernating myocardium, heart failure produces a reversion to a fetal phenotype
of substrate utilization and suppression of adult protein
isoforms with reductions in the activity of members of the
citric acid cycle.17 Lei found PDC to be downregulated at the
transcriptional and protein levels in pacing induced heart
failure.18 Schott demonstrated chamber specific reductions in
PDC and isocitrate dehydrogenase using a proteomic approach in pressure overloaded right ventricles without overt
failure.19 Similarly, Jin reported a downregulation of PDC
and isocitrate dehydrogenase in hypertrophied hearts from
spontaneously hypertensive rats.20 Gallego-Delgado found
NADH dehydrogenase components and cytochrome oxidase
to be reduced in spontaneously hypertensive rats. ATP
synthase ␤ chain subunits were upregulated.21 Faber reported
a downregulation in ATP synthase delta, electron transfer
flavoprotein ␤ subunit and the NADH dehydrogenase
(ubiquinone) ␤ subcomplex.22 Whereas these small animal
models feature cardiac hypertrophy from pressure overload or
failure of the left or right ventricle, similar regional alterations in the expression of regional oxidative enzymes accompany the development of hibernating myocardium.
Regional changes in the expression of cytosolic proteins
such as myoglobin, stress proteins, and cytoskeletal structural
proteins in hibernating myocardium were also similar to the
Page et al
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global alterations reported in advanced heart failure. Myoglobin and creatine kinase M were reduced in hibernating
myocardium as they are in heart failure.23,24 Upregulation in
the expression of stress proteins and antioxidant enzymes
were also similar to heart failure and probably reflect a
general protective mechanism to minimize the production of
reactive oxygen species that ultimately lead to cell death.25
Additional cardioprotective pathways previously identified
using candidate proteins after more severe levels of shortterm repetitive ischemia in swine include GSK-3␤ and H-11
kinase.26,27 The cessation of apoptosis despite persistent
physiological changes of hibernating myocardium suggests
that cardioprotective pathways and downregulation of regional energy utilization may attenuate myocyte loss from
oxidative stress in the chronic state.
Although speculative, the common denominator potentially explaining regional and chamber specificity of the
alteration in oxidative metabolism and stress proteins could
be the development of myocyte cellular hypertrophy. Although there is no evidence of cardiac hypertrophy in terms of
left ventricular mass in swine with hibernating myocardium,
the regional myocyte loss from apoptosis leads to compensatory regional cellular hypertrophy that prevents wall thinning.14 This is further supported by the regional nature of
these changes and the fact that they occur in the absence of
pressure overload or global heart failure. Thus, rather than
resulting from chronic ischemia, the proteomic alterations
reflect the reversion of the hibernating region to regional
cellular hypertrophy.
The upstream transcriptional signaling pathways responsible for the regional mitochondrial protein changes in hibernating myocardium are unknown. The synthesis of metabolic
proteins is regulated by nuclear respiration factors (NRF-1
and NRF-2) and members of the peroxisome proliferator-activated receptor family (PPAR␣, PPAR␤, and PPAR␥).28 –30
NRF-1 and NRF-2 are under the control of the PPAR␥
coactivator-1␣ (PGC-1␣), which can downregulate metabolic
enzymes in response to altered substrate availability. Similarly, because PGC-1␣ is required for activation of PPAR␣ it
could also coordinately downregulate enzymes of fatty acid
oxidation in hibernating myocardium. Further studies will be
required to elucidate the response of these pathways to
ischemia and in hibernating myocardium.
Methodological Limitations
The interpretation of our results needs to consider the cellular
makeup of the whole myocardial tissue sample. Although
myocytes only contribute approximately half of the cells, they
still contribute the majority of protein in a whole tissue
preparation. The total amounts will however be dominated by
the most abundant proteins that include contractile and
mitochondrial proteins. Although this does not diminish the
important regional differences we have reported here, additional studies using subproteomic fractionation will be required to identify less abundant protein changes that may be
“diluted” because of the dominance of contractile and mitochondrial proteins. In addition, differences in myocyte protein content per gram of tissue between hibernating and
normal tissue have been recently reported in a different model
Proteomic Analysis of Hibernating Myocardium
111
by Bito.31 Although Bito showed that calculated mitochondrial volume fraction was increased in hibernating myocardium, we found that mitochondrial protein content per gram
of tissue was similar in the 2 regions. Mitochondrial protein
content was not diminished in hibernating myocardium in
either study. Collectively, these findings support the notion
that reductions in the specific mitochondrial proteins that we
have observed are likely attributable to an adaptive response
whereby the expression of specific genes are selectively
downregulated, and that the reductions are not simply from a
decrease in mitochondrial mass or numbers.
An additional limitation of our approach is the inability to
identify specific posttranslational modifications (PTMs) that
may account for some protein spots. Although mitochondrial
proteins were generally reduced in hibernating myocardium,
there were frequently directionally opposing changes in
contractile proteins that had the same protein identity established by MALDI-TOF. More advanced approaches such as
LC/MS-MS separation will be required to characterize important PTMs. Furthermore, the discovery based proteomic
analysis we used identifies changes in protein expression but
does not examine what effect chronic ischemia has on protein
activity. Although we have demonstrated reductions in activity of specific mitochondrial enzymes, as is the case with
PDC, the cascade of alterations found in hibernating myocardium would likely result in more profound changes in the in
vitro activity for many other proteins, especially multisubunit complexes. Nevertheless, the attenuated metabolic
response and the cessation of apoptosis over time in hibernating myocardium provide integrated in vivo end points
supporting the biological relevance of the protein changes
identified.
Clinical Implications
Our study demonstrates regional reductions in the expression
of mitochondrial oxidative enzymes and other proteins in
hibernating myocardium that are similar to those that occur
globally in hypertrophy, fetal heart, and the advanced failing
heart. These may represent a common response of the
myocyte to stress or cellular hypertrophy resulting in an
attenuation of regional oxygen demand as external workload
is increased at the expense of contractile function. We
speculate that by limiting the development of a supply/
demand imbalance, oxidative stress is minimized and further
myocyte apoptosis and progressive degeneration is prevented.
The reversibility of these proteomic alterations is currently
unknown and has considerable implications for understanding the mechanisms responsible for viable dysfunctional
myocardium in ischemic cardiomyopathy where function can
be severely reduced in the absence of ischemia or fibrosis. If
this reflects a phenotype secondary to compensatory cellular
hypertrophy from apoptosis-induced myocyte loss, it could
explain persistent contractile dysfunction after revascularization. Indeed, almost 1 in 4 dysfunctional segments without
fibrosis by Gd MRI enhancement fail to improve after
coronary revascularization.32 Experiments to evaluate the
effects of revascularization and manipulate the proteomic
changes independently of perfusion will be required to test
this possibility.
112
Circulation Research
January 4/18, 2008
Acknowledgments
We thank Anne Coe, Deanna Gretka, Elaine Granica, and Amy
Johnson for technical assistance.
Sources of Funding
This work was supported by grants from the Department of Veterans
Affairs, the American Heart Association, NHLBI (HL-55324, HL61610), the Albert and Elizabeth Rekate Fund and the John R. Oishei
Foundation.
Disclosures
None.
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Persistent Regional Downregulation in Mitochondrial Enzymes and Upregulation of Stress
Proteins in Swine With Chronic Hibernating Myocardium
Brian Page, Rebeccah Young, Vijay Iyer, Gen Suzuki, Maciej Lis, Lioubov Korotchkina,
Mulchand S. Patel, Kenneth M. Blumenthal, James A. Fallavollita and John M. Canty, Jr
Circ Res. 2008;102:103-112; originally published online October 25, 2007;
doi: 10.1161/CIRCRESAHA.107.155895
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2007 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2007/10/25/CIRCRESAHA.107.155895.DC1
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CIRCRESAHA/2007/155895 R2
ONLINE SUPPLEMENTAL METHODS AND RESULTS
Persistent Regional Downregulation in Mitochondrial Enzymes and Upregulation of Stress
Proteins in Swine with Chronic Hibernating Myocardium
Brian Page
Rebeccah Young
Vijay Iyer
Gen Suzuki
Maciej Lis
Lioubov G. Korotchkina
Mulchand S. Patel
Kenneth M. Blumenthal
James A. Fallavollita
John M. Canty, Jr.
From the VA WNY Health Care System, the Center for Research in Cardiovascular Medicine
and the Center for Excellence in Bioinformatics and Life Sciences at the University at Buffalo.
Abbreviated Title – Proteomic Analysis of Hibernating Myocardium
Correspondence:
John M. Canty, Jr., M.D.
Division of Cardiovascular Medicine
University at Buffalo
Biomedical Research Building, Room 347
3435 Main St.
Buffalo, NY 14214
Phone: 716-829-2663
[email protected]
Page 1
CIRCRESAHA/2007/155895 R2
Supplemental Materials and Methods
Procedures and protocols conformed to institutional guidelines for the care and use of
animals in research. Hibernating myocardium was produced as previously described1. Briefly,
pigs were sedated (Telazol; tiletamine 50mg/ml and zolazepam 50 mg/ml)/xylazine (100 mg/ml,
0.022 mg/kg i.m.) intubated and ventilated with a 0.5–2% isoflurane-oxygen mixture. Through a
limited pericardiotomy, the proximal LAD was instrumented with a Delrin occluder (1.5 mm).
Antibiotics (cefazolin, 25 mg/kg and gentamicin, 3 mg/kg i.m.) were given 1-hour before surgery
and repeated after closing the chest. Analgesia included an intercostal nerve block (0.5%
Marcaine) and prn intramuscular doses of butorphanol (2.2 mg/kg i.m. q6h) and flenixin (1-2
mg/kg i.m. q.d.).
Swine were fed a diet of hog feed pellets (Agway, Minneapolis, MN) consisting of 14%
crude protein, 0.6% lysine, and 3% crude fat. The pellets were produced from grain products,
plant protein products, processed grain by-products, molasses products, and vitamin and mineral
supplements.
Serial Physiological Studies
Physiological studies were conducted 3-months (n=8) or 5-months after instrumentation.
We have previously demonstrated that reductions in flow, function and flow reserve remain
unchanged after 3-months in this model2. Sedation in the closed-chest state was initiated with
Telazol/xylazine and maintained with propofol (5-10 mg/kg/hr i.v.). Under sterile conditions, we
inserted a 6-Fr introducer into the left brachial artery. A multipurpose catheter or Millar end-hole
catheter was inserted into the LV apex for microsphere injection and pressure measurement. The
introducer side port was used to monitor aortic pressure and perform blood withdrawal for
microspheres. Animals were heparinized (100 U/kg), and hemodynamics allowed to equilibrate
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CIRCRESAHA/2007/155895 R2
for at least 30-minutes. Regional wall-thickening was assessed with transthoracic
echocardiography from a right parasternal approach3. All showed anterior dysfunction but
dyskinesis was not present under any condition. Systolic wall-thickening (∆WT=ESWTEDWT) was measured in LAD and remote regions. This was followed by LV microsphere
injection to assess resting perfusion. Pharmacological vasodilation was produced using
adenosine (0.9mg/kg/min iv) with phenylephrine infused and titrated to maintain mean blood
pressure ~100 mmHg and microsphere flow repeated. At the end of the study, catheters were
removed and pigs were brought back to the animal facility.
Animals were allowed to recover before tissue harvesting to circumvent potential
transient effects of pharmacological stimulation secondary to agents used in the physiological
study. After at least 72 hours pigs were brought back to the laboratory and rapidly euthanized
with an injection of KCl after insuring a surgical plane of anesthesia. The hearts were
immediately excised for protein sampling and microsphere flow analysis. The LV was weighed
and sectioned into 1-cm rings parallel to the AV groove from apex to base. Thin rings above
each major ring were incubated in TTC to assess infarction. Subendocardial samples (inner third
of the wall) immediately adjacent to the LAD and posterior descending arteries were flash frozen
for proteomic analyses. Connective tissue was quantified by point counting of trichrome stained
sections, as we have previously described1.
Analysis of Microsphere Flow
Regional perfusion was assessed using 15µm microspheres labeled with fluorescing
dyes3. We injected ~3x106 microspheres into the LV while a reference sample was withdrawn at
6 ml/min for 90-seconds. At the end of the study, samples were taken from a midventricular
ring, divided into twelve circumferential wedges (Figure 1) with each cut into 3 transmural
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CIRCRESAHA/2007/155895 R2
layers. Dyes were extracted using standard techniques and fluorescence quantified at selected
excitation wavelengths4, 5.
Protein Extraction
Tissue from the left ventricle was cut into 3 concentric rings, each of which was divided
into approximately 12 wedges. Wedges were further subdivided into 3 transmural layers. One
ring was used for flow analysis, one for histology, and one for protein sampling. Tissue samples
for proteomic analysis were taken from a subendocardial wedge adjacent to the left anterior
descending coronary artery in the hibernating region (LAD), while remote samples were
obtained from a subendocardial wedge adjacent to the posterior descending artery. Excised
regions were flash frozen in liquid nitrogen and stored at -80° C. Total protein was extracted
from approximately 0.2 g of myocardium by polytron homogenization in 8M urea/2%
CHAPS/2% ß-mercaptoethanol. The homogenate was placed in a sonicating water bath,
centrifuged, and further purified by precipitation with chloroform/methanol.
Pooled sham samples for the 3-month comparison were prepared by adding equal
amounts of protein from four 3-month sham animals, while the pooled sham sample for the 5month comparison was comprised of equal amounts of protein from ten 5-month sham swine.
Differential In Gel Electrophoresis (DIGE)
For identification and quantitation of changes in protein expression, we used the
Differential In Gel Electrophoresis (DIGE) technique.6, 7 Prior to separation by isoelectric
focusing, pharmalytes (pH 3-10NL) were added to the samples to a concentration of 1%,
dithiothreitol (DTT) was added to a final concentration of 65mM, and rehydration buffer [8M
urea/4% CHAPS/1% Pharmalytes (pH 3-10NL)/13mM DTT] was added to a total volume of
450µl. The samples were applied to immobilized pH gradient (IPG) strips (24cm, pH 3-10
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CIRCRESAHA/2007/155895 R2
nonlinear), allowed to absorb by active rehydration, and focused on an IPGphor isoelectric
focusing system.
Strips were then equilibrated for 15 minutes in 6M urea/ 50mM Tris pH 8.8/ 30%
glycerol/ 2% SDS/ 5mg/ml DTT followed by 15 minutes in 6M urea/ 50mM Tris pH 8.8/ 30%
glycerol/ 2% SDS/ 45mg/ml iodoacetamide. Proteins were separated by molecular weight on
12.5% gels in an Ettan DALT SDS-PAGE system (GE Healthcare).
Spot analysis was performed by DeCyder BVA software from GE Healthcare. A paired
student’s t-test was performed on spots present in at least 50% of the gels for changes in volume
ratio, and a p value of ≤ 0.05 was considered statistically significant. For the unpaired analyses,
we manually extracted intensity data on spots present in at least half the gels and calculated an
average volume ratio and an unpaired student’s t-test derived p value for each spot, with a p <
0.05 considered significant. Note that average volume ratios for decreasing spot intensities are
represented in DeCyder as a number less than -1 as the software takes the negative reciprocal of
the average volume ratio. These were converted back to a simple ratio for our analyses, and are
represented as a number between 0 and 1. Experimental pI and MW values were also calculated
by the DeCyder software.
MALDI-TOF and Protein Identification
For in gel digestion, gel pieces were washed for 30 minutes to 1 hour in 100-200 µl of
200 mM ammonium bicarbonate and 50% acetonitrile at 37° C, then dried under vacuum in
either a SpeedVac evaporator, or a desiccator. Trypsin (Promega, Madison, WI) solution (20
µg/ml) was added to the gel pieces, and they were allowed to rehydrate for 20 minutes at room
temperature. Gel pieces were allowed to incubate overnight at 37° C in 40 µl of 40 mM
ammonium bicarbonate /10% acetonitrile. Digests were concentrated and purified with ZipTips
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CIRCRESAHA/2007/155895 R2
(Millipore, Bedford, MA), eluted in approx 1.0 µl of a saturated solution of α-cyano-4hydroxycinnamic acid in 50% acetonitrile in 0.1% trifluoroacetic acid, and spotted on a ground
steel MALDI-TOF target plate.
Additionally, some gel pieces were digested using the automated Proteineer Digest and
Prep Station (Proteineer DP) from Bruker Daltonics (Billerica, MA, USA). The instrument was
run with default settings using digestion reagents provided in Proteineer DP specific chemical
kits. Samples were spotted directly by the instrument onto a 400 µm, 384 sample capacity
Anchor Chip (Bruker Daltonics).
Spectra were obtained using a Bruker Daltonics Biflex MALDI-TOF mass spectrometer
(Bruker Daltonics, Bellerica, MA USA). The spectra in the range of 500-3200 Da were obtained
in reflector mode by the summation of 50-200 laser shots. Peaks were identified and labeled
manually. External calibration of peptide masses was achieved through use of a standard peptide
mix available from Bruker Daltonics. MALDI-TOF-TOF data was obtained using a Bruker
Daltonics Autoflex MALDI-TOF instrument in TOF-TOF mode.
Proteins were identified by peptide mass fingerprinting using Mascot
(www.matrixscience.com). Mascot search parameters included mammalian taxonomy,
carbamidomethyl fixed modification, variable methionine oxidation, 0-2 missed cleavages, and
20-200 ppm mass error. Both the NCBI and Swiss Protein databases were searched. Mascot
MOWSE scores greater than 67 were significant for NCBI, while scores of greater than 61 were
significant for the Swiss Protein database (p ≤ 0.05). The reported scores and accession numbers
are from results of searches conducted through the Swiss Protein database unless otherwise
specified. For MALDI-TOF-TOF identified proteins, MSDB was the chosen search database,
and a score of 39 or higher was considered significant. Theoretical pI and MW values were
Page 6
CIRCRESAHA/2007/155895 R2
obtained from www.expasy.org. For proteins identified as a precursor, the transit sequence was
ignored in the calculation.
Pyruvate Dehydrogenase Complex (PDC) Activity Assays
PDC activity was measured by production of 14CO2 from [1-14C]pyruvate as previously
described.8 Frozen tissue samples from the LAD (hibernating) and remote (normal) areas of eight
3-month animals were homogenized in MOPS-KCl buffer (50 mM MOPS buffer, pH 7.4
containing 80 mM KCl, 2 mM MgCl2, 0.5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride
and 0.5 µg/ml leupeptin). Homogenates were freeze/thawed three times in liquid nitrogen to
break up mitochondria. Supernatant was separated by centrifugation at 600x g for 10 min. For
measurement of ‘active’ PDC activity, aliquots of homogenates were incubated in MOPS-KCl
buffer containing 4.4 mM dichloroacetate and 30 mM NaF to inhibit both pyruvate
dehydrogenase kinase and pyruvate dehydrogenase phosphatase, respectively. For measurement
of ‘total’ PDC activity, aliquots of treated homogenates (100 µg) were incubated with pyruvate
dehydrogenase phosphatase 1 (10 µg) in MOPS-KCl buffer, (containing 10 mM MgCl2, 1 mM
CaCl2, and 4.4 mM dichloroacetate to inhibit pyruvate dehydrogenase kinase) for 30 min at 30°
C to fully dephosphorylate and activate the enzyme complex.
PDC reaction was performed in the presence of 2 mM MgCl2, 2 mM NAD+, 15 mM
potassium oxalate, 0.8 mM DTT, 0.1 mM TPP, 0.64 mM CoA in 50 mM potassium phosphate
buffer, pH 7.5 at 37° C, and the reaction was started with 0.5 mM [1-14C]pyruvate. Reaction
tubes were immediately sealed with rubber stoppers having center wells with paper filters
presoaked with benzetonium hydroxide to absorb released 14CO2. Reaction was stopped after 10
min, and tubes were incubated for 1 h to collect liberated 14CO2. Radioactivity was measured
with a scintillation counter.
Page 7
CIRCRESAHA/2007/155895 R2
A 50mM stock solution of radioactive pyruvate, with specific radioactivity of 2000
cpm/nmol, was used in the reaction. The solution was comprised of dissolved lyophilized
radioactive [1-14C]pyruvate and non-radioactive pyruvate. Specific radioactivity of pyruvate was
calculated based on the total pyruvate concentration (radioactive pyruvate + unlabeled pyruvate).
Enzyme activity is expressed as nmol per min per mg of total cellular protein.
Dihydrolipoamide dehydrogenase (E3) activity was measured spectrophotometrically by
formation of NADH in the forward reaction9. Aliquots of homogenates (50 µl) were incubated
with 150 µl of hypotonic solution (20 mM potassium phosphate, pH 7.4, containing 1% Triton
X-100, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin) on ice for 30
min and centrifuged for 10 min at 14,000 g. Supernatants were used to measure enzyme activity.
The reaction mixture contained 100 mM potassium phosphate, pH 8.0, 1.5 mM EDTA, 3 mM
NAD+ and 3 mM dihydrolipoamide. Reaction was started by adding treated homogenate (15-25
µg of total cellular protein). Activity is expressed as µmol of NADH per minute per mg of total
cellular protein.
Cytochrome C Oxidase and Citrate Synthase Activity Assays
Cytochrome c oxidase and citrate synthase activities were measured
spectrophotometrically on total protein from frozen tissue using commercial kits available from
Sigma.10, 11 Animal tissue selected for activity included nine of the ten animals used for the 5
month proteomics, and ten of the eleven 5 month shams that were used in the proteomic analysis.
Frozen samples stored in a -80° C freezer were thawed, weighed, and minced using a McIlwain
tissue chopper. Tissue pieces were washed in 2 volumes of extraction buffer (containing 100
mM MOPS, 550 mM KCl, 5 mM EGTA at ph 7.5), and treated with 10 volumes of 0.25 mg/ml
trypsin (dissolved in extraction buffer) initially for 3 minutes, followed by 8 volumes for 20
Page 8
CIRCRESAHA/2007/155895 R2
minutes, to dissolve away connective tissue and myofibrils and aid in the release of
mitochondria. The trypsin was neutralized with 50 mg/ml of albumin in water added to a final
concentration of 10 mg/ml, the tissue pieces were again washed with extraction buffer, and
subsequently homogenized with a polytron homogenizer. Homogenates were centrifuged at
600x g for 8 minutes and an aliquot of the supernatant was collected as the total protein fraction.
Total protein preps were assayed for protein concentration using the DC Protein Assay
from Bio-Rad. Reactions were carried out in a 1 ml cuvette and absorbance measured on an
Hitachi U-2000 UV/vis spectrophotometer using a time scan as per the instructions in the Sigma
assay kits. All reagents needed for reaction were provided in the Sigma kits. Total protein
samples for cytochrome c oxidase were placed in enzyme dilution buffer (250 mM sucrose and
10 mM Tris-HCl at pH 7.5) containing 1 mM n-dodecyl ß-D-maltoside, while those for citrate
synthase were placed in CelLytic M Cell Lysis Reagent (Sigma), to disrupt mitochondrial
membranes and release the enzymes into solution. Cytochrome c oxidase activity was calculated
based upon the difference between extinction coefficients of 21.84 for reduced and oxidized
ferrocytochrome c at 550 nm12, where 1 unit of activity represents 1 µmole of ferrocytochrome c
oxidized per minute at pH 7 at room temperature. Citrate synthase activity was calculated based
upon the extinction coefficient of 13.6 of 5-thio-2-nitrobenzoic acid at 412 nm. Baseline
reaction was carried out to account for any endogenous levels of thiol or deacetylase activity, and
subtracted from the activity observed after addition of oxaloacetate. Units are expressed in
µmoles/ml/min.
To analyze mitochondrial protein content from frozen tissue; remaining supernatants
were transferred to a new tube and subsequently spun at 11,000x g for 12 minutes to obtain a
Page 9
CIRCRESAHA/2007/155895 R2
pellet enriched with mitochondria. Pellets were resuspended in equal volumes, and
mitochondrial protein concentration was assayed using the Bio-Rad DC Protein Assay.
Statistics
Data are expressed as mean±standard error. Comparisons between hibernating and remote
normally perfused regions of the same heart were assessed using paired t-tests. To evaluate for
differences in myocardial perfusion, a two-way ANOVA (p<0.05) was used with a post-hoc
paired t-test. For evaluation of differences between animals with hibernating myocardium and
age matched sham control groups, and for serial changes in protein expression in hibernating
myocardium evaluated 3 and 5-months after instrumentation, we used unpaired t-tests.
Supplemental Results
Hemodynamics in 3 and 5-month Swine with Hibernating Myocardium
At the time of study, heart rate was 94 ± 7 bpm in the 3 month group versus 98.3 ± 6 bpm
in the 5 month group, left ventricular end-diastolic pressure averaged 23.3 ± 1.6 mm Hg in the 3
month group versus 22.4 ± 2.4 in the 5 month group, while aortic systolic and diastolic pressures
averaged 147 ± 7 mm Hg and 89 ± 4 mm Hg, respectively in the 3 month group versus 130 ± 6
mm Hg and 83 ± 6 mm Hg, respectively in the 5 month group. There were no significant
differences between experimental groups.
Proteomic Quantitation and Spot Identification
Supplemental table 1 lists the characteristics of identified spots. These include
experimental pI and MW values calculated for each identified spot from the master gel,
theoretical pI and MW values as determined by www.expasy.org, protein name, number of
Page 10
CIRCRESAHA/2007/155895 R2
peptides matched, % sequence coverage, Mascot MOWSE score, species from which the Mascot
match was made, existence of a published pig sequence, and Swiss Protein accession number.
We identified 52 differentially expressed protein spots representing 37 unique proteins. An
additional 62 spots have been identified which were not significantly altered representing 35
unique control proteins. For all identified spots, the average Mascot Mowse score for each
protein was 99 with 25% average peptide sequence coverage. Of the 72 unique proteins
identified, 35 had known porcine sequences.
Regional protein expression in sham animals with normal myocardium
Regional differences in protein expression in 3 month sham animals with normal
myocardium were minimal (Supplemental table 2). The large majority of identified spots
demonstrated no significant difference in abundance in the LAD territory relative to the remote
(PDA) territory from sham animals. The only exceptions were pyruvate dehydrogenase alpha
subunit (9%), mitofilin (12%), creatine kinase M chain (5%), and myosin heavy chain beta
(28%). With the exception of myosin heavy chain beta, the magnitude of the change was 12% or
less, and in many cases, there were additional spots identified as the same protein which did not
demonstrate a significant difference in regional expression.
Comparison of LAD/remote and LAD/sham protein abundance ratios in 3 month animals
with hibernating myocardium
Differences between LAD/remote and LAD/sham measurements in 3 month animals with
hibernating myocardium were also minimal (Supplemental table 2). When comparing the ratio
of a given protein spot from the LAD territory of 3 month animals with hibernating myocardium
to that from the remote territory of the same animals, or to the pooled (LAD and remote) samples
from 3 month sham animals, the differences were largely similar with the magnitude of the
Page 11
CIRCRESAHA/2007/155895 R2
change often greater in the LAD/sham comparison. Because of use of unpaired comparisons,
many of the changes in the LAD/sham group did not reach statistical significance despite having
a greater difference in ratio of protein abundance. Notable proteins where the LAD/sham ratio
differed markedly from the LAD/remote ratio included mitochondrial creatine kinase, NADP+
isocitrate dehydrogenase 47 kDa subunit, and myosin heavy chain beta. Mitochondrial creatine
kinase and NADP+ isocitrate dehydrogenase 47 kDa subunit were relatively equally expressed in
the LAD and remote regions of 3 month animals with hibernating myocardium, however when
the LAD expression was compared to a pooled sham sample, the ratio was significantly reduced.
This may be explained by remote zone remodeling whereby the remote territory of 3 month
animals with hibernating myocardium may undergo some of the same protein expression
changes as in the hibernating region such that the resulting LAD/remote protein abundance ratio
becomes normalized. In the case of mitochondrial creatine kinase and NADP+ isocitrate
dehydrogenase 47 kDa subunit, the data support that these proteins are likely commensurately
reduced in the LAD and remote regions of 3 month animals with hibernating myocardium.
Temporal stability of changes in protein expression at 3 and 5 months
Of the 114 spots identified, 25 showed significant differences in LAD/sham protein
abundance ratio at 5-months in comparison with swine studied early after the development of
hibernating myocardium (3-months). Mitochondrial protein expression remained largely
downregulated at 5-months with notable exceptions including long chain Acyl-CoA
dehydrogenase, ATP synthase, mitochondrial CK, and mitochondrial aspartate aminotransferase,
all of which returned towards normal at 5-months. Dihydrolipoamide succinyltransferase and
the flavoprotein subunit of complex II of the electron transport chain were further downregulated
at 5-months relative to 3-months. Aconitase consisted of two protein spots, one of which
Page 12
CIRCRESAHA/2007/155895 R2
increased at 5-months while the other decreased. Similarly, some contractile proteins also were
significantly downregulated at 5-months relative to 3-months, while others were upregulated.
Pyruvate kinase became significantly downregulated at 5-months as did triosephosphate
isomerase. Increases in cytoskeletal proteins at 3-months normalized after 5-months, while
stress proteins such as αB crystallin, HSP2β6 and HSP-27 remained chronically elevated in
hibernating myocardium. Supplemental Figure 1 summarizes the regional and temporal
alterations of mitochondrial proteins, Supplemental Figure 2 summarizes the results for
cytoskeletal proteins early and late after the development of hibernating myocardium, and
Supplemental Figure 3 summarizes the changes in abundance in contractile proteins at 3 and 5
months.
Correlation of Proteomic Changes with Physiological Parameters of Hibernating
Myocardium
In an effort to explore the physiological significance of the down-regulated proteins in
hibernating myocardium, regression analysis was performed against LVEDP, regional wall
thickening, and relative subendocardial perfusion at rest and during adenosine vasodilation
(LAD/remote) for all chronically instrumented animals (3 and 5 months after instrumentation).
The LAD/Normal protein expression ratios for fifteen proteins were evaluated including the
downregulated proteins summarized in the text, as well as mitochondrial aspartate
aminotransferase, cytoplasmic malate dehydrogenase (involved in the malate-aspartate shuttle),
and troponin T. There was no significant correlation between physiological changes and the
relative protein changes except for 4 proteins (data not shown). The two most closely correlated
relationships were for ATP Synthase α versus relative subendocardial flow under resting
conditions (Supplemental Figure 4a, r2=0.45, p=0.003), and troponin T versus relative
Page 13
CIRCRESAHA/2007/155895 R2
subendocardial flow during adenosine vasodilation (Supplemental Figure 4b, r2=0.48,
p=0.001).
Page 14
CIRCRESAHA/2007/155895 R2
Supplemental Figure Legends
Supplemental Figure 1 – Summary of Mitochondrial Protein Expression at 3 and 5months. The majority of idenitifed mitochondrial proteins were downregulated at 3 –months and
are represented by blue boxes. These include citric acid cycle, electron transport, and ATP
synthesis enzymes. Prohibitin, mitofilin, and medium chain acyl-CoA dehydrogenase were
unchanged and are represented by uncolored boxes. Aconitase and dihydrolipoamide
succinyltransferase were upregulated as represented by yellow boxes. Hatched boxes represent
those proteins that were differentially altered at 5-months. These were often associated with a
trend toward normalization at 5-months.
Supplemental Figure 2 – Summary of Cytosolic Protein Expression at 3 and 5-months.
Many cytosolic proteins remained unchanged in expression at 3-months, including GAPDH,
DRP-2, and aminoacylase-1, and are represented by uncolored boxes. Upregulated proteins are
represented by yellow boxes, and included predominantly stress and structural proteins.
Downregulated proteins, represented by blue boxes, included myoglobin, lactate dehydrogenase
L chain, malate dehydrogenase, cytoplasmic aspartate aminotransferase, and pyruvate kinase.
Proteins differentially altered at 5 months are represented by a hatched box. At 5-months, some
glycolytic enzymes became downregulated, while structural proteins tended to decrease in
expression to normal.
Supplemental Figure 3 – Summary of Contractile Protein Expression at 3 and 5-months. At
3-months, actin, troponin, myosin light chain 1, and tropomysin ß chain were downregulated
(represented by blue boxes), while myosin heavy chain α and tropomysin α chain were
Page 15
CIRCRESAHA/2007/155895 R2
unchanged in expression (represented by uncolored boxes). Myosin heavy chain ß was
upregulated at 3-months as represented by the yellow box. Hatched boxes represent those
contractile proteins differentially altered at 5-months relative to 3-months.
Supplemental Figure 4a – Linear Regression Analysis of LAD/Normal protein expression
ratio for ATP Synthase α against relative subendocardial resting flow. Protein expression of
ATP Synthase α in the hibernating region demonstrated a direct relationship with relative
subendocardial resting flow which was strongest in the 3 and 5 month combined group.
Supplemental Figure 4b - Linear Regression Analysis of LAD/Normal protein expression
ratio for Troponin T against relative adenosine vasodilated flow. Protein expression of
Troponin T in the hibernating region demonstrated a direct relationship with relative
subendocardial adenosine flow which was strongest in the 3 and 5 month combined group.
Page 16
CIRCRESAHA/2007/155895 R2
Supplemental References
1.
Fallavollita JA, Perry BJ, Canty JM, Jr. 18F-2-deoxyglucose deposition and regional flow
in pigs with chronically dysfunctional myocardium: Evidence for transmural variations
in chronic hibernating myocardium. Circulation. 1997;95:1900-1909.
2.
Fallavollita JA, Logue M, Canty JM, Jr. Stability of hibernating myocardium in pigs with
a chronic left anterior descending coronary artery stenosis: Absence of progressive
fibrosis in the setting of stable reductions in flow, function and coronary flow reserve. J
Am Coll Cardiol. 2001;37:1989-1995.
3.
Malm BJ, Suzuki G, Canty JM, Jr., Fallavollita JA. Variability of contractile reserve in
hibernating myocardium: Dependence on the method of stimulation. Cardiovasc Res.
2002;56:422-433.
4.
Glenny RW, Bernard S, Brinkley M. Validation of fluorescent-labeled microspheres for
measurement of regional organ perfusion. J Appl Physiol. 1993;74:2585-2597.
5.
Thomas SA, Fallavollita JA, Borgers M, Canty JM, Jr. Dissociation of regional
adaptations to ischemia and global myolysis in an accelerated swine model of chronic
hibernating myocardium. Circ Res. 2002;91:970-977.
6.
Unlu M, Morgan ME, Minden JS. Difference gel electrophoresis: a single gel method for
detecting changes in protein extracts. Electrophoresis. 1997;18:2071-2077.
7.
Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F, Hawkins E,
Currie I, Davison M. Validation and development of fluorescence two-dimensional
differential gel electrophoresis proteomics technology. Proteomics. 2001;1:377-396.
Page 17
CIRCRESAHA/2007/155895 R2
8.
Kerr DS, Ho L, Berlin CM, Lanoue KF, Towfighi J, Hoppel CL, Lusk MM, Gondek CM,
Patel MS. Systemic deficiency of the first component of the pyruvate dehydrogenase
complex. Pediatr Res. 1987;22:312-318.
9.
Patel M, Hong Y. Lipoic acid as an antioxidant: The role of dihydrolipoamide
dehydrogenase. In: Armstrong D, ed. Free Radical and Antioxidant Protocols. Totowa,
NJ: Humana Press Inc.; 1998:337-346.
10.
Storrie B, Madden EA. Isolation of subcellular organelles. In: Deutscher MP, ed.
Methods in Enzymology. Guide to Protein Purification. Vol 182. Philadelphia: Elseiver;
1990:203-225.
11.
Srere PA. Citrate synthase. In: Lowenstein JM, ed. Methods in Enzymology. Citric Acid
Cycle. Vol 13. Philadelphia: Elsevier; 1969:3-11.
12.
Berry EA, Trumpower EL. Simultaneous determination of hemes a, b, and c from
pyridine hemochrome spectra. Anal Biochem. 1987;161:1-15.
Page 18
CIRCRESAHA/2007/155895 R2
Supplemental Table 1 – Protein Identification with MALDI-TOF
Theor
Spot Theor MW
Exp
ID
pI
(Da)
pI
Mitochondrial Proteins
Citric Acid Cycle Proteins
Decreased
111
7.69
82761 7.43
119
7.69
82761
7.6
120
7.69
82761 7.69
162
5.51
59652 5.47
340
6.31
50198 6.76
344
6.31
50198 6.95
415
5.89
41528 5.86
871
8.55
33083 8.39
872
8.55
33083 8.55
870
8.55
33083 8.77
748
5.71
36653 5.87
767
5.71
36653 6.07
850
5.38
36107 5.41
476
6.51
40286 7.12
Exp
MW
(Da)
Protein
75407
74866
74866
70672
57448
57138
52954
31989
31931
31874
37014
36419
33043
48674
aconitase
aconitase
aconitase
pyruvate dehydrogenase complex E2 subunit
dihydrolipoamide dehydrogenase (E3)
dihydrolipoamide dehydrogenase (E3)
dihydrolipoamide succinyltransferase
malate dehydrogenase (mitochondrial)
malate dehydrogenase (mitochondrial)
malate dehydrogenase (mitochondrial)
NAD+ isocitrate dehydrogenase (α subunit)
NAD+ isocitrate dehydrogenase (α subunit)
pyruvate dehydrogenase E1 ß subunit
pyruvate dehydrogenase E1 α subunit
Increased
151
7.69
433
5.89
72608
51469
aconitase
dihydrolipoamide succinyltransferase
82761
41528
7.44
5.87
Peptides
matched
Electron Transport Chain and ATP Synthesis Proteins
Decreased
438
5
51563 4.88 53222 ATP synthase F1 ß chain
431
4.95
51710 4.93 53222 ATP synthase F1 ß chain
382
8.27
55263 8.18 55017 ATP synthase F1 α chain
386
8.27
55263 8.34 54918 ATP synthase F1 α chain
Page 19
%
cov
Score
species
Pig
ID
Accession
15
12
11
9
5
5
11
4
6
4
4
7
8
9
22%
22%
20%
15%
13%
15%
20%
19%
26%
17%
11%
28%
34%
20%
91
124
110
73
71
85
91
68
84
80
70
92
150
78
pig
pig
pig
pig
human
pig
pig
pig
pig
pig
monkey
orangutan
pig
pig
yes
yes
yes
yes†
yes
yes
yes
yes
yes
yes
no
no
yes†
yes
P16276
P16276
P16277
gi|47522814
P09622
P09623
Q9N0F1
P00346
P00346
P00346
Q28480
Q5R678
gi|448581
P29804
8
9
12%
18%
81
128
cow
pig
yes
yes
P16276
Q9N0F1
10
20
10
10
23%
40%
26%
26%
116
153
68
100
cow
cow
human
cow
no
no
no
no
P00829
P00829
P25705
P19483
CIRCRESAHA/2007/155895 R2
384
387
403
1173
1052
427
429
135
138
471
213
8.27
8.27
8.27
5.71
5.45
7.08
7.53
5.24
5.24
5.46
6.25
55263
55263
55263
23760
30207
48499
48505
77183
77183
49212
68012
8.25
8.46
6.59
5.94
5.53
7.44
7.58
5.27
5.32
5.28
6.67
54918
54819
53937
18366
22792
52122
51841
73133
73001
48849
66954
ATP synthase F1 α chain
ATP synthase F1 α chain
ATP synthase F1 α chain
Complex I - 24 kDa subunit
Complex I - 30 kDa subunit
Complex I - 51 kDa subunit
Complex I - 51 kDa subunit
Complex I - 75 kDa subunit
Complex I - 75 kDa subunit
Cytochrome Bc1 core protein I - Complex III
Flavoprotein subunit of Complex II
8
16
15
8
5
11
5
5
MS/MS
6
8
20%
35%
36%
34%
19%
37%
16%
8%
24%
16%
100
131
149
82
74
97
72
70
49
76
95
cow
cow
cow
human
mouse
cow
human
mouse
human
cow
human
no
no
no
no
no
no
no
no
no
no
no
P19483
P19483
P19483
P19404
Q9DCT2
P25708
P49821
Q91VD9
P28331
P31800
P31040
Other Mitochondrial Membrane Proteins
Inner Membrane
Decreased
985
5.57
29804 5.45 26619 prohibitin
122
6.08
83678 6.26 74866 mitofilin
6
8
28%
11%
103
67
human
human
no
no
P35232
Q16891
Increased
99
6.08
83678
5.99
76640
mitofilin
9
12%
70
human
no
Q16891
Outer Membrane
Decreased
934
8.62
30726
932
8.62
30726
924
7.5
31592
8.42
8.74
7.62
30923
29711
30197
voltage-dependent anion channel 1 (VDAC1)
voltage-dependent anion channel 1 (VDAC1)
voltage-dependent anion channel 2 (VDAC2)
4
5
9
25%
30%
42%
94
94
131
pig
pig
human
yes
yes
yes
Q9MZ16
Q9MZ16
Q9MZ15
8
13
8
5
28%
31%
22%
13%
94
126
102
65
yes
no
no
no
P00506
P17540
P17540
P26440
5
MS/MS
4
10%
67
50
77
pig
human
human
human
mouse,
chi hmster
pig
pig
no
yes
yes
P38647
P79274
P41367
Other Mitochondrial Matrix Proteins
Decreased
695
8.98
44664 9.02 39423
510
7.28
43360 7.86 46951
543
7.28
43360 7.69 46196
542
6.9
43069 7.23 45371
165
621
613
5.5
6.2
8.1
68612
44707
43384
5.37
6.8
6.71
70672
44707
43373
Aspartate aminotransferase (mitochondrial)
creatine kinase (mitochondrial)
creatine kinase (mitochondrial)
Isovaleryl-CoA Dehydrogenase
Mitochondrial Stress-70 protein (GRP 75)
long chain Acyl-CoA dehydrogenase (LCAD)
medium chain Acyl-CoA dehydrogenase
Page 20
12%
CIRCRESAHA/2007/155895 R2
46629
46629
52230
45077
8.4
8.47
7.02
6.79
45290
46784
52230
46867
NADP+ isocitrate dehydrogenase (47 kDa subunit)
NADP+ isocitrate dehydrogenase (47 kDa subunit)
Succinyl-CoA 3-ketoacid-CoA Transferase 1
translation elongation factor EF-Tu
Cytoplasmic Proteins
Glycolysis Proteins
Decreased
491
6.81
46893
646
8.4
39211
642
8.4
39211
487
7.73
46855
298
7.95
57805
1109
7.09
26581
7.88
8.6
8.42
7.7
7.83
7.36
48237
41613
41613
48324
59770
20611
ß enolase
fructose biphosphate aldolase A
fructose biphosphate aldolase A
muscle specific phosphopyruvate hydratase
pyruvate kinase
triosephosphate isomerase
Increased
451
6.99
782
8.52
786
8.52
788
8.52
47037
35704
35704
35704
6.82
8.34
8.57
8.84
50097
35898
35769
35833
α enolase
GAPDH
GAPDH
GAPDH
1063
28634
7.21
21795
phosphoglycerate mutase 2 M isozyme
Contractile Proteins
Decreased
641
5.23
42051
604
5.23
42018
1313
5.23
42018
634
5.23
42051
630
5.23
42018
54
5.63 223097
394
5.64 223083
1143
5.03
21800
1189
5.03
21800
775
4.66
32850
859
4.71
32694
693
6.08
31482
5.03
5.45
6.32
5.15
5.21
5.34
5.51
5
4.99
4.64
4.94
5.19
41613
43373
7570
41914
42293
81775
54425
19040
17844
35963
33301
39924
actin alpha cardiac
actin alpha cardiac
actin alpha cardiac
actin alpha skeletal
actin alpha skeletal
myosin heavy chain ß
myosin heavy chain ß
myosin light chain 1
myosin light chain 1
tropomysin ß chain
tropomysin α chain
Troponin T (cardiac isoform 2)
564
568
338
509
8.5
8.5
6.67
6.2
9
Page 21
13
9
5
8
33%
23%
14%
20%
130
130
90
80
pig
pig
pig
cow
yes
yes
yes
no
P33198
P33198
Q29551
P49410
6
8
8
5
7
5
19%
35%
31%
21%
13%
22%
61
96
90
75
64
79
mouse
rabbit
rabbit
human
rabbit
mouse
no
no
no
no
no
yes
P21550
P00883
P00883
P13929
P11974
P17751
MS/MS
4
7
4
21%
27%
12%
43
66
96
64
no
yes
yes
yes
P06733
P00355
P00355
P00356
11
58%
136
multiple
pig
pig
pig
human,
mouse
no
P18669
7
6
5
9
9
24
8
8
4
11
11
MS/MS
20%
20%
13%
29%
28%
15%
14%
37%
23%
32%
30%
97
66
71
125
111
179
63
100
74
152
121
129
human
multiple
human
cow
human
human
rat
human
human
human
rat
multiple
no
no
no
yes
yes
no*
no*
no
no
no
yes
no
P68032
P68032
P68032
P68138
P68133
P12883
P02564
P08590
P08590
P07951
P04692
Q8CH03
CIRCRESAHA/2007/155895 R2
719
717
737
5.92
5.37
5.92
31243
33633
33783
5.22
5.27
5.38
38372
38580
37755
Troponin T cardiac
Troponin T cardiac
Troponin T cardiac
4
5
6
16%
22%
17%
58
86
65
human
sheep
human
no
no
no
P45379
P50751
P45379
Increased
663
5.23
1106
5.23
745
5.23
390
5.4
409
5.64
5
5.63
461
5.6
42018
42018
42051
54264
223083
223097
223689
5.74
5.37
5.43
5.6
5.45
5.06
5.75
41091
20500
37148
54622
53454
93268
49648
actin alpha cardiac
actin alpha cardiac
actin alpha skeletal
myosin heavy chain ß
myosin heavy chain ß
myosin heavy chain ß
myosin heavy chain α
5
5
5
11
23
14
18
18%
15%
22%
23%
12%
8%
11%
84
63
84
103
112
92
76
human
human
human
cat
pig
human
human
no
no
yes
no*
no*
no*
no
P68032
P68032
P68133
gi|12656128
P79293
P12883
P13533
Structural Proteins
Increased
224
6.17
66193
333
5.21
53497
406
5.21
53497
407
5.21
53497
398
5.21
53497
366
5.06
53520
21
5.62 123813
6.77
5.26
5.07
5.16
5.23
5.01
6.11
66473
57344
53743
53743
54035
55815
86627
actin interacting protein
desmin
desmin
desmin
desmin
vimentin
vinculin
5
5
11
15
11
9
13
13%
15%
29%
38%
35%
24%
20%
75
84
108
146
184
72
140
human
pig
pig
pig
pig
human
pig
no
yes
yes
yes
yes
no
yes
O75083
P02540
P02540
P02540
P02540
P08670
P26234
Heat Shock Related Proteins
Decreased
6.86, 22248,
1236 7.39
20854
7.72
181
5.53
70083 5.22
182
5.53
70083 5.28
291
5.91
60955 5.21
15702
69912
69661
60420
α B crystallin, Phosphatidylethanolamine-binding
protein (PEBP)
HSP 70 (70 Kda protein 1)
HSP 70 (70 Kda protein 1)
HSP60
6, 5
6
7
6
37%,
36%
13%
21%
14%
74,
62
66
94
65
sheep,
cow
human
human
mouse
yes,
no
yes
yes
no
Q5ENY9,
P13696
P11142
P11142
P63038
Increased
1219
6.76
1237
6.76
154
5.01
155
5.01
1128
6.23
16249
15505
71827
71827
19739
α B crystallin
α B crystallin
GRP78
GRP78
HSP 27
13
10
7
12
7
61%
54%
16%
21%
36%
180
135
112
129
108
rabbit
rabbit
human
human
pig
yes
yes
no*
no*
yes
P41316
P41316
P11021
P11021
Q5S1U1
20129
20129
70478
70478
22942
7.43
7.42
4.87
4.91
6.6
Page 22
CIRCRESAHA/2007/155895 R2
1125
1133
1238
6.23
6.23
5.95
22942
22942
17135
5.51
5.56
6.13
19810
19491
15092
HSP 27
HSP 27
HSP20 beta-6
6
6
3
32%
28%
15%
97
86
62
pig
pig
human
yes
yes
yes
Q5S1U1
Q5S1U1
O14558
Antioxidant Proteins
Increased
1222
4.7
14168
1295
6.04
15760
5.01
6.24
16278
9097
Peroxiredoxin-2
superoxide dismutase
4
5
36%
46%
84
100
pig
pig
yes
yes
P52552
P04178
Other Cytoplasmic Proteins
Decreased
659
6.8
46343 7.53
609
6.63
42971 7.01
614
6.77
43101 7.19
619
6.77
43101 7.35
830
5.58
36481 5.46
846
6.15
36323
6.2
1271
6.83
16953
7.5
41240
43766
43373
42907
33644
33222
11727
Aspartate aminotransferase (cytoplasmic)
creatine kinase, M chain (cytoplasmic)
creatine kinase, M chain (cytoplasmic)
creatine kinase, M chain (cytoplasmic)
L-lactate dehydrogenase B chain
malate dehydrogenase (cytoplasmic)
myoglobin
18
5
5
15
7
13
9
49%
17%
16%
37%
24%
44%
63%
226
75
64
180
109
184
151
pig
pig
human
human
pig
pig
pig
yes
yes
yes
yes
yes
yes
yes
P00503
Q5XLD3
P06732
P06732
P00336
P11708
P02189
Increased
79
6.23
640
5.62
247
5.95
349
6.02
16%
15%
20%
49
96
76
89
human
pig
mouse
human
no
yes
no
no
P21399
P37111
O08553
P78371
87
pig
yes
P08835
202
146
105
93
275
pig
pig
pig
pig
pig
yes
yes
yes
yes
yes
P08835
P08835
P08835
P02067
P09571
98398
45216
62293
57357
6.49
5.71
6.31
6.33
79023
41763
64854
56830
aconitase 1 (Fe regulatory protein 1)
Aminoacylase-1
dihydropyrimidinase related protein 2 (DRP-2)
T-complex protein 1 (chaperonin)
MS/MS
6
5
8
Other Proteins
Serum Proteins
Decreased
212
5.84
66797
6.39
67074
albumin
MS/MS
Increased
203
5.84
202
5.84
423
5.84
1319
7.25
144
6.93
5.95
5.8
5.73
7.57
7.03
67438
67803
52029
7210
72738
albumin
albumin
albumin
hemoglobin ß subunit
serotransferrin
66797
66797
66797
16034
76967
16
17
10
9
23
Page 23
38%
38%
22%
60%
43%
CIRCRESAHA/2007/155895 R2
Endoplasmic Reticulum Proteins
290
5.61
54265 5.81 60420
protein disulfide isomerase A3
9
19%
99
human
no
P30101
Other Proteins
777
7.53
38545
808
6.9
38481
annexin 2
annexin 2
6
9
24%
24%
91
76
mouse
cow
no
no
P07356
P04272
7.35
7.35
36028
34941
*only a fragment of the pig gene has been sequenced
† pig sequence found only in the NCBI database. Reported Mascot search statistics and accession number are from the NCBI database
MS/MS = identification made by MALDI-TOF-TOF data and MS/MS ion search feature of Mascot
Page 24
CIRCRESAHA/2007/155895 R2
Supplemental Table 2 – Quantitation of Protein Changes from 2D-DIGE
3 month
Sham LAD
vs. Remote
Av ratio n=
Spot ID Protein
Mitochondrial Proteins
Citric Acid Cycle Proteins
Decreased
111 aconitase
119 aconitase
120 aconitase
162 pyruvate dehydrogenase complex E2 subunit
340 dihydrolipoamide dehydrogenase (E3)
344 dihydrolipoamide dehydrogenase (E3)
415 dihydrolipoamide succinyltransferase
871 malate dehydrogenase (mitochondrial)
872 malate dehydrogenase (mitochondrial)
870 malate dehydrogenase (mitochondrial)
748 NAD+ isocitrate dehydrogenase (α subunit)
767 NAD+ isocitrate dehydrogenase (α subunit)
850 pyruvate dehydrogenase E1 ß subunit
476 pyruvate dehydrogenase E1 α subunit
3 month
Hibernating
LAD vs.
Remote
Av ratio n=
3 month
Hibernating
LAD vs.
Sham
Av ratio n=
5 month
Hibernating
LAD vs.
Sham
Av ratio n=
1.04
1.04
1.06
0.98
0.94
0.96
1.01
1.03
1.03
1.03
1.01
0.98
0.97
1.09*
7
7
7
7
7
6
7
7
7
7
7
7
7
7
0.88
0.79
0.83
0.8
0.94
0.89
0.92
0.78*
0.71*
0.79*
0.72*
0.85*
0.74*
0.83
8
7
7
8
8
8
5
6
8
7
8
8
8
8
0.88
0.82
0.77
0.72
0.85
0.79
1.04
0.74
0.77
0.74
0.58*
0.72*
0.64*
0.64*
7
7
7
8
8
8
4
8
8
8
8
8
8
8
1.22†
1.10
0.92
0.68*
0.81
N/A
0.80†
0.78
0.85
0.76
0.66*
0.71*
0.58*
0.63*
10
9
10
10
10
Increased
151 aconitase
433 dihydrolipoamide succinyltransferase
1.04
0.98
7
2
1.32*
1.42*
7
7
0.91
1.19
8
8
0.55†
1.19
7
8
Electron Transport Chain and ATP Synthesis Proteins
Decreased
438 ATP synthase F1 ß chain
431 ATP synthase F1 ß chain
382 ATP synthase F1 α chain
386 ATP synthase F1 α chain
0.94
0.98
1.01
1.02
7
7
5
6
0.73*
0.75*
0.79*
0.79*
8
8
7
7
0.55*
0.56*
0.52
0.76
8
8
4
6
0.78†
0.75*†
0.84†
N/A
10
10
7
Page 25
8
3
10
10
10
10
10
10
CIRCRESAHA/2007/155895 R2
384
387
403
1173
1052
427
429
135
138
471
213
ATP synthase F1 α chain
ATP synthase F1 α chain
ATP synthase F1 α chain
Complex I - 24 kDa subunit
Complex I - 30 kDa subunit
Complex I - 51 kDa subunit
Complex I - 51 kDa subunit
Complex I - 75 kDa subunit
Complex I - 75 kDa subunit
Cytochrome Bc1 core protein I - Complex III
Flavoprotein subunit of Complex II
1.01
1.06
0.93
0.98
1.00
0.96
1.02
1.02
1.01
0.93
1.00
4
6
7
7
7
7
7
7
7
7
7
0.75*
0.79*
0.76*
0.83*
0.86*
0.83*
0.8*
0.88*
0.8
0.82*
0.86*
8
7
8
8
8
8
8
7
8
8
8
0.62*
0.79
0.73
0.73*
0.75
0.92
0.84
0.88
0.55
0.77*
0.89
7
8
7
6
7
8
8
4
6
8
7
0.83†
0.81
0.72
0.76*
0.58*
0.78*
0.82
0.91
0.78
0.71
0.60*†
9
10
10
10
10
10
10
10
10
10
10
Other Mitochondrial Membrane Proteins
Inner Membrane
Decreased
985 prohibitin
122 mitofilin
0.98
1.11
7
7
0.84
0.85
6
7
0.72
0.98
8
7
0.96†
0.79
6
6
Increased
99 mitofilin
1.12*
7
1.22
6
1.44
6
1.01†
10
Outer Membrane
Decreased
934 voltage-dependent anion channel 1 (VDAC1)
932 voltage-dependent anion channel 1 (VDAC1)
924 voltage-dependent anion channel 2 (VDAC2)
1.03
1.00
0.98
7
7
7
0.84*
0.86*
0.89*
8
8
8
0.89
0.81
0.68
8
8
8
0.94
0.89
0.79
7
10
9
Other Mitochondrial Matrix Proteins
Decreased
695 Aspartate aminotransferase (mitochondrial)
510 creatine kinase (mitochondrial)
543 creatine kinase (mitochondrial)
542 Isovaleryl-CoA Dehydrogenase
165 Mitochondrial Stress-70 protein (GRP 75)
621 long chain Acyl-CoA dehydrogenase (LCAD)
613 medium chain Acyl-CoA dehydrogenase
564 NADP+ isocitrate dehydrogenase (47 kDa subunit)
1.07
1.06
1.01
0.98
1.01
1.04
1.02
1.04
6
5
6
7
7
7
7
7
0.86*
0.81
1
0.6*
0.91*
0.76*
0.83
0.89
8
6
7
8
8
8
8
8
0.66
0.81
0.66*
0.71
0.87
0.61
0.76
0.59*
7
2
6
5
8
8
8
8
0.98†
0.66*
1.06†
0.70*
0.95
0.89†
0.82
0.58*
5
5
4
10
10
10
10
10
Page 26
CIRCRESAHA/2007/155895 R2
568
338
509
NADP+ isocitrate dehydrogenase (47 kDa subunit)
Succinyl-CoA 3-ketoacid-CoA Transferase 1
translation elongation factor EF-Tu
1.13
0.93
1.01
5
6
7
0.92
0.79
0.79*
6
7
8
0.52*
0.64*
0.68
6
8
8
0.60
0.69*
0.78
9
10
10
Cytoplasmic Proteins
Glycolysis Proteins
Decreased
491 ß enolase
646 fructose biphosphate aldolase A
642 fructose biphosphate aldolase A
487 muscle specific phosphopyruvate hydratase
298 pyruvate kinase
1109 triosephosphate isomerase
1.05
1.04
0.99
1.03
1.02
0.98
7
5
7
7
7
7
0.8
0.76
0.87
0.89
0.85*
0.9
5
5
6
4
7
8
0.96
0.81
0.73
0.94
0.95
0.79
4
3
8
8
7
8
0.89
0.64
0.69*
0.93
0.65*†
1.01†
10
7
10
10
10
10
Increased
451
782
786
788
1063
1.01
0.98
1.01
1.01
1.01
7
6
7
6
7
1.62
1.13
1.1
1.19
1.04
8
7
8
5
8
1.37
1.09
0.99
0.85
1.06
5
7
8
4
8
1.05†
1.07
0.92
1.12
0.70*†
10
10
10
8
10
0.96
1.00
N/A
1.1
1.02
1.28*
1.28
1.00
1.00
1.00
0.96
0.93
0.9
0.9
7
7
0.74*
0.96
1.01
1.01
0.95
0.86
0.97
0.8
0.81*
0.8
0.85
0.82*
0.88*
0.84*
8
8
7
7
8
3
7
8
7
8
8
8
8
8
0.67
1.12
1.13
0.89
0.93
1.94*
1.01
0.61*
0.64*
0.55
0.55
0.69
0.81
0.74*
8
5
3
7
7
7
6
8
8
7
7
5
8
8
0.67*
1.06
N/A
0.58*†
0.42†
1.04
1.44†
N/A
0.52*†
0.57*
0.70†
0.66*
0.67*
0.68*
10
10
α enolase
GAPDH
GAPDH
GAPDH
phosphoglycerate mutase 2 M isozyme
Contractile Proteins
Decreased
641 actin alpha cardiac
604 actin alpha cardiac
1313 actin alpha cardiac
634 actin alpha skeletal
630 actin alpha skeletal
54 myosin heavy chain ß
394 myosin heavy chain ß
1143 myosin light chain 1
1189 myosin light chain 1
775 tropomysin ß chain
859 tropomysin α chain
693 Troponin T (cardiac isoform 2)
719 Troponin T cardiac
717 Troponin T cardiac
Page 27
6
7
7
7
7
6
7
7
6
7
7
10
10
5
9
8
10
10
10
10
10
CIRCRESAHA/2007/155895 R2
737
Troponin T cardiac
0.95
7
0.94
8
0.93
7
0.65*†
10
actin alpha cardiac
actin alpha cardiac
actin alpha skeletal
myosin heavy chain ß
myosin heavy chain ß
myosin heavy chain ß
myosin heavy chain α
1.06
0.94
1.05
1.29*
1.22
1.39
1.05
4
6
6
7
7
4
6
1.39
1.61
2.32
1.1
1.54
1.1
1.7
8
8
8
8
8
4
8
2.05
1.78
2.37
1.17
3.1
1.27
3.17
6
7
8
7
8
3
8
0.71
0.89
0.90
1.21*
1.19
0.73
1.19
5
7
10
1
3
4
8
Structural Proteins
Increased
224 actin interacting protein
333 desmin
406 desmin
407 desmin
398 desmin
366 vimentin
21 vinculin
0.99
1.11
0.93
1.08
1.03
1.12
1.03
7
5
4
6
7
7
7
1.08
1.23
1.41*
1.37
1.62
2.14*
1.63
8
6
7
8
8
8
5
0.88
1.72
1.16
1.70
1.69
2.34
1.19
8
5
6
8
8
8
4
0.87
1.18
0.67
1.23
1.37
1.23†
1.27
10
5
6
10
10
8
10
Heat Shock Related Proteins
Decreased
1236 α B crystallin, Phosphatidylethanolamine-binding protein (PEBP)
181 HSP 70 (70 Kda protein 1)
182 HSP 70 (70 Kda protein 1)
291 HSP60
0.97
1.00
0.99
1.00
7
7
7
7
0.93
0.88
0.93
0.79
8
8
8
8
0.76*
0.91
0.78
0.64*
8
7
8
8
0.72*
1.07
0.89
0.88†
10
10
10
10
Increased
1219
1237
154
155
1128
1125
1133
1238
0.85
0.92
0.9
0.93
0.8*
0.91
0.94
0.71
7
7
7
7
7
2
7
5
2.07*
1.36*
1.54
1.29
1.96*
1.39
1.33
2.55*
8
6
8
8
8
5
7
5
1.67
0.92
1.33
1.16
1.32
0.96
1.01
1.32
7
8
7
8
8
6
7
7
1.57*
0.88
1.25*
1.19*
1.51*
1.21
1.19*
1.64*
10
9
10
10
10
9
1
10
Increased
663
1106
745
390
409
5
461
α B crystallin
α B crystallin
GRP78
GRP78
HSP 27
HSP 27
HSP 27
HSP20 beta-6
Page 28
CIRCRESAHA/2007/155895 R2
Antioxidant Proteins
Increased
1222 Peroxiredoxin-2
1295 superoxide dismutase
1.03
N/A
7
1.22*
1.49*
8
7
1.1
1.44
8
2
0.92
0.20
10
1
Other Cytoplasmic Proteins
Decreased
659 Aspartate aminotransferase (cytoplasmic)
609 creatine kinase, M chain (cytoplasmic)
614 creatine kinase, M chain (cytoplasmic)
619 creatine kinase, M chain (cytoplasmic)
830 L-lactate dehydrogenase B chain
846 malate dehydrogenase (cytoplasmic)
1271 myoglobin
1.03
1.05*
0.98
1.04
1.03
0.98
1.08
7
7
7
7
5
7
7
0.86
0.69*
0.66*
0.75*
0.83
0.8*
0.8*
8
8
8
8
7
8
8
0.67*
0.72*
0.64*
0.67
0.68
0.64*
0.44
8
8
8
8
4
8
7
.54*
0.84
0.69*
0.54*
0.45*
0.64*
0.46*
10
9
10
10
9
10
10
Increased
79
640
247
349
1.00
0.94
0.99
0.97
7
3
7
7
1.14
1.08
1.19
1.01
5
7
8
8
1.02
1.31
1.08
0.95
5
3
8
8
1.00
0.64
0.95
0.88
10
4
9
10
Other Proteins
Serum Proteins
Decreased
212 albumin
0.99
7
0.90*
8
0.79
7
0.86
9
Increased
203
202
423
1319
144
1.01
0.98
1.13
N/A
1.11
6
7
6
8
8
8
5
7
1.08
0.89
2.68*
1.15
1.22
8
6
8
8
6
0.81
0.89
1.76
N/A
0.37*†
9
10
7
7
1.47
1.22
1.67*
1.05
1.38
0.99
7
1.4
8
1.34
8
1.07
10
aconitase 1 (Fe regulatory protein 1)
Aminoacylase-1
dihydropyrimidinase related protein 2 (DRP-2)
T-complex protein 1 (chaperonin)
albumin
albumin
albumin
hemoglobin ß subunit
serotransferrin
Endoplasmic Reticulum Proteins
290 protein disulfide isomerase A3
Page 29
5
CIRCRESAHA/2007/155895 R2
Other Proteins
777 annexin 2
808 annexin 2
0.99
1.07
*
p ≤0.05 vs. remote or sham.
† p <0.05 3 month LAD/sham vs. 5 month LAD/sham
N/A = spot not present
Page 30
7
3
1.75*
1.8*
8
8
1.71*
1.61
8
4
1.10†
1.05†
10
7
Supplemental
Figure 1
Complex I
NADH
dehydrogenase
75 kDa subunit
Complex II
Complex II
Flavoprotein
subunit
Complex III
Cytochrome Bc1
core protein 1
Complex V
ATP Synthase
F1 α chain
Oxidative
Phosphorylation
Inner
mitochondrial
membrane
NADH
dehydrogenase
51 kDa subunit
Mitochondrial
Matrix
Outer
mitochondrial
membrane
NADH
dehydrogenase
24 kDa subunit
ATP Synthase
F1 ß chain
Mitofilin
Protein
Transport/Assembly
Mitochondrial
Proteins
NADH
dehydrogenase
30 kDa subunit
Prohibitin
Pyruvate
dehydrogenase
Pyruvate
dehydrogenase
E1 α subunit
Pyruvate
dehydrogenase
E1 ß subunit
Pyruvate
dehydrogenase
E2
Dihydrolipoamide
dehydrogenase
E3
Citric Acid
Cycle
Aconitase
Isocitrate
dehydrogenase
α subunit
Malate
dehydrogenase
Dihydrolipoamide
succinyltransferase
Beta
Oxidation
Long chain
acyl CoA
dehydrogenase
Medium chain
acyl CoA
dehydrogenase
Metabolite
Catabolism
Isovaleryl CoA
dehydrogenase
Aspartate
aminotransferase
High Energy
Phosphate Metabolism
Voltage
dependent anion
channel 1
Voltage
dependent anion
channel 2
Protein
Synthesis
Cell
Proliferation
and aging
Succinyl CoA 3
ketoacid CoA
transferase 1
Mitochondrial
Creatine kinase
Translation
elongation factor
EF-Tu
NADP+ Isocitrate
dehydrogenase
47 kDa subunit
Mitochondrial
stress protein
70 (GRP 75)
Glycolytic
Enzymes
α enolase
GAPDH
ß – enolase
Phosphoglycerate
mutase 2 M
isozyme
Fructose
bisphosphate
aldolase A
Muscle specific
phosphopyruvate
hydratase
Triosephosphate
isomerase
Structural
Proteins
Vimentin
Heat Shock
Related
Proteins
HSP 70
(70 kDa
Protein 1)
Superoxide
Dismutase
Antioxidant
Proteins
Vinculin
Desmin
GRP 78
HSP 27
Anaerobic
Metabolism
MalateAspartate
Shuttle
HSP 20
beta-6
Pyruvate
Kinase
α B crystallin
HSP 60
Peroxiredoxin 2
High Energy
Phosphate
Metabolism
Cytosolic
Proteins
Oxygen carrying
proteins
Actin
Interacting
Protein
Myoglobin
Cytoplasmic
Creatine
Kinase
M chain
L lactate
dehydrogenase
B chain
Malate
dehydrogenase
Aspartate
Aminotransferase
Chaperones
Dehydropyrimidinases
Aminoacylases
RNA Binding
T-Complex
protein 1
(Chaperonin)
Dihydrpryimidinase
Related protein 2
(DRP-2)
Aminoacylase-1
Aconitase 1
(Fe regulatory
protein 1)
Supplemental
Figure 2
Actin
Cardiac α actin
Myosin
Myosin heavy
chain ß
Troponin
Cardiac
troponin T
Contractile
Proteins
Tropomyosin
Tropomyosin
ß chain
Skeletal α actin
Myosin heavy
chain α
Myosin
light chain 1
Tropomyosin
α chain
Supplemental
Figure 3
ATP Synthase α
1.0
LAD/Normal
0.8
3 months
5 months
3 + 5 Months
Linear (3 months)
Linear (5 months)
Linear (3 + 5 Months)
0.6
R2 = 0.7099
0.4
R2 = 0.2169
R2 = 0.4509
0.2
0.0
0.0
0.2
0.4
0.6
0.8
1.0
Relative (LAD/Remote) Resting
Endocardial Flow
1.2
Supplemental
Figure 4A
Troponin T
1.2
LAD/Normal
1.0
0.8
0.6
3 months
5 months
3 + 5 Months
Linear (3 months)
Linear (5 months)
Linear (3 + 5 Months)
R 2 = 0.145
R 2 = 0.8092
0.4
R 2 = 0.4816
0.2
0.0
0.0
0.1
0.2
0.3
0.4
Relative (LAD/Remote) Adenosine Flow
Supplemental
Figure 4B