Page 1Articles
of 39 in PresS. Am J Physiol Heart Circ Physiol (September 1, 2006). doi:10.1152/ajpheart.00078.2006
Ning et al.
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Short Cycle Hypoxia in the Intact Heart: Hypoxia Inducible Factor 1Signaling and the Relationship to Injury Threshold
Xue-Han Ning1,3,4, Shi-Han Chen2, Norman E. Buroker 1, 3, Cheng-Su Xu4, Fu-Ren Li4, Shu-Ping
Li4, De-Song Song4, Ming Ge3, Outi M. Hyyti1, Min Zhang3 and Michael A. Portman1,3
From the Divisions of Cardiology1 and Genomics and Development2, Department of Pediatrics,
University of Washington, and Children’s Hospital and Regional Medical Center3, Seattle WA
98105; and Qinghai-Tibet High Altitude Physiology Collaborative Group4, Key Laboratory of
Hypoxia Physiology, Chinese Academy of Sciences, Shanghai, China
(Total 4746 words)
Short Title: HIF in short-cycle hypoxia
This study was supported in part by the grants from Children’s Hospital and Regional Medical
Center (HR5836), NSFC 38970307, and NIH (HL60666).
Address for correspondence:
Michael A. Portman MD
Cardiology MS W4841
Children’s Hospital and Regional Medical Center
4800 Sand Point Way NE
Seattle WA, 98105
206-987-5153
[email protected]
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Copyright © 2006 by the American Physiological Society.
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Abstract
Hypoxia inducible factor1 (HIF-1 ) transcriptionally activates multiple genes, which regulate
metabolic cardioprotective and cross-adaptive mechanisms. Hypoxia and several other stimuli
induce the HIF-1 signaling cascade, though little data exist regarding the stress threshold for
activation in heart. We tested the hypothesis that relatively mild short-cycle hypoxia, which
produces minimal cardiac dysfunction and no sustained or major disruption in energy state, can
induce HIF-1 activation. We developed a short-cycle hypoxia protocol in isolated perfused rabbit
heart to test this hypothesis. By altering cycling conditions, we identified a specific cycle with
O2 content and duration, which operated near a threshold for causing functional injury in these
rabbit hearts. Mild short-cycle hypoxia for 46 minutes elevated HIF-1 mRNA and protein
within 45 minutes after reoxygenation. Expression also increased for multiple HIF-1 target
genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1(Hmox1). After
mild hypoxia, VEGF protein accumulation occurred, although HIF-1 and VEGF protein
accumulation were suppressed after more severe hypoxia, which also caused depletion of ATP
and non-diffusible nucleotides. In summary, these results indicate that mild near-threshold
hypoxia induces HIF-1 cascade, but more severe hypoxia suppresses protein accumulation for
this transcription factor and the target genes. Posttranscriptional suppression of these proteins
occurs under conditions of altered energy state, exemplified by ATP depletion.
Keywords: ATP, Heme oxygenase, Oxygen consumption, Reoxygenation, VEGF
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Introduction
Hypoxia induces adaptive responses in heart, which promote resistance to other
environmental stressors (28). This cross-tolerance is due to the activation of common protective
pathways including induction or reprogramming of gene signaling pathways and modification of
posttranscriptional mechanisms (14). Cross-tolerance may provide a therapeutic mode of
reducing effects of noxious stimuli, such as ischemia during cardiac surgery.
Many of these adaptive responses are mediated by hypoxia inducible factor 1 (HIF-1 ).
Oxygen concentration influences HIF-1 by multiple mechanisms including posttranslational
modifications, nuclear translocation, heterodimerization with the HIF-1ß-subunit, and target gene
trans-activation (4). A decrease in ATP concentration or production, generally accompanying
hypoxia, putatively triggers some of these mechanisms (14). However, oxygen independent
pathways, such as hormones, cytokinines, and growth factors can all trigger accumulation of
HIF-1 and its transcriptional activation. HIF-1 also responds to reactive oxygen species and
nitric oxide signaling. In the intact heart, HIF-1 activation and induction have been studied
either after prolonged heat stress (30 days), severe intermittent hypoxia (2) or after ischemia with
myocardial infarction (14). Furthermore, HIF-1 contributes to the mechanism for cross
tolerance between heat shock and ischemia. Although these chronic forms of stress induce HIF1 and promote HIF-1 protein stabilization, they would provide limited clinical benefit because
of their severe and prolonged nature.
The principal objective of this study was to determine, if mild acute hypoxic stress
induces a modification in HIF-1 signaling. We therefore designed an experiment, where we
first determined the cardiac functional threshold for oxygen deprivation using short-cycle
hypoxia. The cycle durations used in this study have direct relevance to clinical scenarios as well
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as high altitude medicine. For instance, cycling (15-90 seconds) in arterial oxygen saturation
occurs during nocturnal periodic breathing induced by high altitude (4500-8000 meters) (9, 16,
23). Similarly, patients with congestive heart failure exhibit 60-second cycles during sleep (8,
12). Using the short-cycle hypoxia protocol, we determined if the HIF-1 mRNA and protein
response occur at O2 deprivation levels, which do not surpass the injury threshold, and if the
response was divorced from major metabolic or functional disturbance.
Methods
Preparation of Isolated Hearts
Rabbit hearts (male or female, 2.3-2.8 kg body weight; anesthetized with sodium
pentobarbital 45 mg/kg and heparinized with 700 U/kg, intravenously) were rapidly excised and
momentarily immersed in ice-cold physiological salt solution (PSS). Procedures followed were
in accordance with the Guide for the Care and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No.85-23, revised 1996). The preparation has
been reported previously (17). In brief, the aorta was cannulated using the Langendorff mode
perfused with PSS equilibrated with 95% O2-5% CO2 at 37°C and pH 7.4. The PSS contained
118.0 mmol/L NaCl, 4.0 mmol/L KCl, 22.3 mmol/L NaHCO3, 11.1 mmol/L glucose, 0.66
mmol/L KH2PO4, 1.23 mmol/L MgCl2, and 2.38 mmol/L CaCl2 and was passed twice through
filters with 3.0 Km pore size. Perfusion pressure was maintained at 90 mmHg (17, 18). A
pressure transducer was connected to a balloon, which was placed in the left ventricle through
the mitral orifice to measure left ventricular pressure (LVP) and its first derivative with respect to
time (dP/dt). The balloon volumes were varied over a range of values to construct left
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ventricular Pressure-Volume curves to define an optimal function condition with developed
pressure between 100 and 140 mmHg and end-diastolic pressures less than 8 mmHg at baseline.
This volume remained unchanged during the entire experiment. The coronary flow was
measured with a flow meter (T201, Transonic Systems Inc., Ithaca, NY) that was connected to a
cannula in the pulmonary artery. Myocardial oxygen consumption (MVO2) was calculated as
MVO2 = coronary flow × [(PaO2-PvO2) × (c/760)]/Vm, where CF is coronary flow (ml/min/g wet
weight); (PaO2-PvO2) is the difference in the partial pressure of oxygen (PO2, mmHg) between the
coronary affluent and effluent; c is the Bunsen solubility coefficient of O2 in perfusate at 37°C
(22.7 Kl O2 × atm-1×ml-1 perfusate), and Vm is the molar volume (22.4 ml/mmol). Lactate
production was the difference between coronary effluent and coronary affluent concentration
times coronary flow. The analog signals were recorded with an online computer (Macintosh) and
an analog signal acquisition system (Biopac System, Inc., Santa Barbara, CA) and a pressurized
ink chart recorder (Gould Inc., Cleveland, OH). The developed pressure (DP) was defined as
peak systolic pressure (PSP) minus end-diastolic pressure (EDP). The weight of the heart was
determined at the conclusion of each experiment after trimming the great vessels and fat and blot
drying with nine-layer cotton gauze. Baseline data were obtained after 30 minutes equilibration
period. During the baseline period, data were obtained with the hearts maintained at 37°C by
circulating water at this temperature through the wall of organ bath.
Short Cycle Protocol
Short-cycle hypoxia was induced by timed alteration of hypoxic PSS infusion with
normal oxygenated PSS (19Kl/ml). The hypoxic PSS O2 content was varied by mixing PSS
bubbled with 95% Nitrogen and 5% CO2 mixture gas with oxygenated PSS. We used a 120
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second full cycle containing hypoxia and reoxygenation components, and exposed each heart to a
single variation of this cycle with respect to hypoxia duration. Accordingly, hearts underwent
one of the following hypoxia/reoxygenation (H/R) protocols: 10sec/110sec, 30sec/90 sec, and
60sec/60sec. Preliminary data showed that at least 60sec is required for recovery within each
individual cycle. The H/R duration and the cycle intervals for this study were based on clinically
relevant hypoxia cycling documented at high altitude (16, 23) and during congestive heart failure
(12). Forty-two hearts were used in this entire study. Most underwent the short-cycle hypoxia
protocols defined above. Functional analyses for four or more hearts were evaluated at each time
or intensity point. Twelve hearts were perfused under control conditions (C) without hypoxic
exposure.
Two other groups (A & S) were defined in the first stage of experiments (shown in
results) by their cardiac function response to short-cycle hypoxia and their relationship to the
functional injury threshold. In this study, subthreshold is defined as oxygen deprivation
inadequate to elicit a decrease in cardiac function. Accordingly, the oxygen content would be
higher than oxygen content that would surpass the threshold. Group A (H/R 10sec/110sec) was
defined as receiving subthreshold doses of oxygen deprivation, while group S (H/R 60sec/60sec)
received hypoxic exposure, which surpassed the injury threshold. Both A and S hearts were
perfused with O2 content of 2.2 µl/ml for 23 cycles. Samples for energy metabolites were
collected after 15 minutes of reoxygenation following completion of the 23rd cycle or at the
comparable time in controls, group C.
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Energy metabolite measurements
Hearts were rapidly frozen with tongs and stored in liquid N2. Tissue was lyophilized for
48 hours at -40oC under 200-torr vacuum. An aliquot (10 mg) of the dried tissue was
homogenized with 800 Kl of 0.73 M trichloroacetic acid (TCA). After centrifugation (7,000 rpm,
2 min) at 4oC, the supernatant (400 Kl) was removed and added to a new Eppendorf tube
containing an equal volume of tri-n-octylamine and Freon (1:1, v/v). The sample mixture was
then vortexed and centrifuged as before. The aqueous phase was analyzed by high-performance
liquid chromatography (HPLC) with Waters 484 ultraviolet absorbance detector for nucleotide
(ATP, ADP, AMP, IMP) and nucleoside (hypoxanthine, xanthine, adenosine, inosine) (20, 21).
Analysis was performed with Waters Maxima 820 software and NEC Power Mate 1.
RNA Isolation
Total RNA was extracted with a RNA Isolation Kit (Ambion Inc., Austin, TX) from an
aliquot (100mg) of pulverized, homogenized frozen tissue. RNA samples were tested by
ultraviolet absorption at A260 nm to determine the concentration and the quality was further
confirmed by electrophoresis on denatured 1% agarose gels (22, 24).
Northern Blot Analysis
Fifteen Kg of RNA were denatured and electrophoresed in a 1% formaldehyde agarose
gel, transferred to a nitrocellulose transfer membrane (Micron Separations Inc., Westboro, MA),
and cross-linked to the membrane with short-wave ultraviolet cross linker. The hybridizing
solutions contained 50% formamide, 1X Denhardt’s solution, 6X SSPE, and 1% sodium dodecyl
sulfate (SDS). The heat shock protein (HSP) 70-1 mRNA levels were detected using a 1.7 kb
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cDNA fragment cloned from human hippocampus (ATCC, Rockville, MD)(17, 19). The
glucose regulated protein 94 (Grp94) mRNA levels were detected using a 427 bp cDNA
fragment cloned from rabbit heart (made by our laboratory). The complementary DNA (cDNA)
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probes were labeled with [ P]dCTP by random primer extension (PRIME-IT II, Stratagene, La
Jolla, CA) and added to the hybridizing solution to a specific activity. Hybridization was carried
out at 42°C for 18 hours followed by several blot washings with a final wash in 1X standard
sodium citrate (SSC) and 0.1% SDS at 65°C. The blots were exposed on PhosphorImager
(Model 400S, Molecular Dynamics, Sunnyvale, CA) and Kodak Biomax MS film (Eastman
Kodak Co., Rochester, NY) at -70°C. The RNA loading was normalized by comparison to that
of glyceraldehydes-3-phosphate dehydrogenase (GAPDH)(17, 19). To compare different mRNA
levels in the same myocardial sample, mRNAs were analyzed by means of sequentially reprobing
the membranes for GAPDH, HSP70-1, and Grp94 cDNA probes (sequenced in our laboratory).
Hypoxia Microarray Analysis
A hypoxia signaling pathway cDNA microarray was used to compare gene expression
between rabbit heart samples taken from the three groups. Microarrays were used to identify
genes with potential for response to short cycle hypoxia. The GEArray Q series gene expression
array contains 96 genes presumably regulated by hypoxia (Super Array Bioscience Corporation,
Frederick, MD). Total RNA from four heart samples with in each group were pooled to provide
an aliquot of three micrograms per group that could be used to make Biotin-16-dUTP labeled
cDNA, which was then used as a probe of gene expression on the microarray. The labeled cDNA
from each group was hybridized overnight to a hypoxia microarray. The next day the
microarrays were washed to remove any unhybrized probe. Chemiluminescence was used to
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detect gene expressed and the results were recorded on Kodak BioMax Light-1 film. All
procedural steps in the GEArray expression array kit provided by the supplier were followed.
Specific array signal spots were analyzed with ImageQuant quantitation software (Molecular
Dynamics, Sunnyvale, CA) (18).
Immunoblotting
Specific proteins were selected for content determination, based primarily on results
from the hypoxia microarray. Fifty micrograms of total protein extracts from rabbit heart tissue
were electrophoresed along with one lane containing thirty micrograms of human HeLa cells as a
positive control and one lane of molecular weight size markers (chemichrome western control,
Sigma) in 4.5% stacking and either a 7.5, 10 or 12% running SDS-polyacrylamide gels. The gels
were then electroblotted onto PDVF plus membranes. The western blots were blocked for one
hour at room temperature with 5% non-fat milk in Tris-buffered saline plus Tween-20
(TBST)[10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20], followed by overnight
incubation at 4°C with each primary antibody diluted in the appropriate blocking solution as
recommended by the supplier. The primary antibodies glucose transporter-4 (Glut4, 400064) and
glucose regulated protein-94 (Grp94, 368675) were obtained from Calbiochem, EMD
Biosciences, Inc. The primary antibodies Il6st (gp130, sc-656), heme oxygenase 1 (Hmox1, sc1797), heat shock protein 70 (HSP70, sc-1060), hypoxia-inducible factor (HIF-1 , sc12542), peroxisomal proliferator activated receptor (PPAR) gamma coactivator-1 (PGC-1, sc5816), and vascular endothelial growth factor (VEGF, sc-152) were obtained from Santa Cruz
Biotechnology. After two 5- minute washes with TBST and one 5- minute wash with Tris-
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buffered saline (TBS), membranes were incubated at room temperature for one hour with the
appropriated secondary antibody conjugated to horseradish peroxidase (HRP). The membranes
were washed twice for 10 minutes with TBST and visualized with enhanced chemiluminescence
after exposure to Kodak biomax light ML-1 film. The membranes were stripped by washing
them 2 times for 30 minutes with 200 mM Glycine, 0.1% SDS and 1% Tween-20 (pH adjusted to
2.2), followed by three 10-minute washes with TBS. The membranes were again blocked for one
hour as above, followed by overnight incubation at 4°C with a -Actin antibody (Santa Cruz
Biotech, sc-1616) diluted 1:200 in blocking solution. The next day the membranes were washed
(as above), the appropriate secondary-HRP antibody was applied, and the remaining procedures
as described above were followed. The -actin was used to verify protein lane loadings.
Statistical Analysis
The reported values are means ± standard error (SE) in the text, tables, and figures. The
S-PLUS Version 6.0 Program (Insightful corp., Seattle, WA, 2005) was used for statistical
analysis. Data were evaluated with repeated measure analyses of variance within groups and
single factor analysis of variance across groups. When significant F values were obtained,
individual group means were tested for differences. The criterion for significance was P < 0.05
for all comparisons.
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Results
Hypoxia threshold
In order to determine hypoxia threshold, we needed to define a set value for contractile
function impairment. Our preliminary studies indicated that we could confidently resolve a 10%
decrease in left ventricular developed pressure. Therefore, LVDP < 90% baseline was defined as
functional impairment.
Developed pressure shows recovery to less than 90% baseline during reoxygenation after
23 cycles (Figure 1). Thus, this particular heart displayed contractile impairment after the shortcycle hypoxia protocol. To determine the hypoxia intensity threshold, the hearts were exposed to
a given exposed duration and cycles, 10-second hypoxia followed by 110 sec reoxygenation and
total of 23 cycles. The hearts displayed no deviation from baseline developed pressure
throughout the protocol until O2 content in the perfusate decreased to below 12 Kl/ml for 5 cycles
from the normal O2 content of 19 Kl/ml. This response was transient for hearts exposed to 7.4,
4.6, 2.4, and 2.2 µl/ml for 23 cycles (46 min), and full recovery was achieved rapidly with
reoxygenation for each cycle. Cycling with an O2 content U 2.0 Kl/ml impaired functional
recovery during the cycle’s reoxygenation portion, thereby defining O2 content between 2.0 - 2.2
Kl/ml as the injury oxygen threshold for these cycling conditions (Figure 2).
Cycle-length and number
The next phase was to define the number of cycles operating at previously defined O2
content (2.2 Kl/ml), which would reasonably maintain cardiac function, and presumably energy
status. The principal goal was to achieve at least 45 minutes of stability and allow a 45-minute
recovery period prior to sampling for metabolic state and mRNAs (see discussion). Perfusing at
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O2 content at 2.2Kl/ml maintained cardiac function for as long as 23 cycles (46 minutes).
Extending the hypoxia cycle duration to 30 seconds impaired contractile function during the
reoxygenation phase (Figure 3). Doubling the hypoxia cycle duration (60 seconds) did not
exacerbate functional impairment, implying that O2 content and not hypoxia duration within the
cycle determined functional impairment, once threshold was surpassed (Figure 3). The short
duration of the hypoxia period in the H/R 10/110 (Group A) and 30/90 cycles precluded accurate
sampling of steady-state oxygen consumption during the hypoxic and recovery portion of these
cycles. Details for cardiac function, coronary flow and myocardial oxygen consumption during
cycling and recovery are shown in Table 1 for the hypoxia duration of 60 seconds and for the
control group. Data were obtained for each portion of the first cycle, hypoxia (H1) and
reoxygenation (R1), and for the final, 23rd cycle, H23 and R23. Significant differences (P < 0.05)
between these groups occur for most of these parameters by the end of the 23rd hypoxic cycle.
However, reoxygenation abrogates many of these differences implying lack of profound
persistent impairment caused even by H/R 60/60sec (group S).
The relative coronary flow responses during the hypoxic portions for the first and twentythird cycles in groups C, A, and S are shown in Figure 4. Coronary flow did not increase in A
compared to C during the hypoxic portions of the cycling periods, though the potential for
increasing coronary flow exists as shown by the data for group S receiving the 60/60sec protocol
(Table 1 and Figure 4). The dramatic coronary flow response in S (160±2.0%) demonstrates that
C and A have not accessed their full coronary reserve. The coronary flow returned to baseline
during the reoxygenation portion of the cycles. High coronary flow apparent in S distinguishes
this model from ischemic variants, and provides a mechanism for energy metabolite washout
(discussed later). For all cycle lengths with O2 content beyond the injury intensity threshold,
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function progressively deteriorated as cycle number increased (Figure 3), though not significantly
until reoxygenation cycle 10. The progressive deterioration due to increasing cycle number
indicates that cycle effect is additive once intensity threshold is surpassed.
Energy metabolism and metabolic stress
Myocardial ATP, total non-diffusible nucleotides (TNN) and total diffusible nucleosides
(TDN) were measured for three heart perfusion conditions (Figure 5). ATP and TNN were not
noticeably different between C and A, but significantly decreased in S compared to C and A (P <
0.05). The concentrations of TDN were not statistically different between the groups. Minimal
lactate production occurred during control conditions and cycling in the A group (Figure 6).
However, S showed substantial increases in lactate production throughout the protocol.
Gene expression during mild and severe short-cycle hypoxia
We analyzed mRNA expression in three groups C, A, and S, as previously defined. We
compared the control group, C, with a single group exhibiting metabolic stress and functional
injury (S), and a group exhibiting no significant ATP depletion and functional injury (A), but
exposed to hypoxic cycling. Samples were extracted from hearts after 45 minutes reoxygenation
following completion of 23 cycles. Our initial Northern blot analyses revealed that expression
for two stress-related genes HSP70 and Grp94 relative to GAPDH were significantly altered by
both mild and more severe hypoxia (Figure 7). GAPDH mRNA content relative to -tubulin was
not different among the groups (data not shown). As Grp94 is putatively regulated by HIF-1 ,
we then performed more extensive analyses of the HIF-1 signaling pathway using the hypoxia
microarray in order to identify target genes and their proteins, which might show responses
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during our protocol. We defined change in gene expression by comparisons among groups
relative to GAPDH and actin as indicated in Figure 8. As array samples were pooled, and
therefore could not be subjected to statistical analyses, we used strict criteria for defining
differences in expression. Upregulation in A or S groups was defined as fold change greater than
2.0 compared to C, and down regulation less than 0.6. The HIF-1 signal within the chips
relative to signal for many genes was relatively low. Nevertheless, we measured fold changes by
our criteria in both A and S relative to C. Table 2 shows results for known HIF-1 target genes.
Also shown are results for other important hypoxia regulated genes, which demonstrated
differences among groups. All other genes on the microarray chip are included in the
supplemental table 1. These showed no change in expression among the groups. The HIF-1
target gene response to these protocols was highly variable as short-cycle hypoxia induced
vascular endothelial growth factor (VEGF), glucose transporter 1 (Glut1), and glucose
transporter 2, but did not alter expression for erythropoietin (EPO). Genes related to other
signaling pathways are also included on the chip. For example the apoptosis inhibitor, survivin
(BIRC5) is downregulated by severe short-cycle hypoxia.
Protein expression response to short-cycle hypoxia
Immunoblots revealed a significant increase in HIF-1 and VEGF protein in group A
compared to control (Figure 9). Although a modest increase in these proteins also occurred in
group S, differences from C did not reach significance. Relative expression for eight proteins is
included in Table 3. The selection of proteins for these immunoblot experiments were based on
positive results from the microarray and availability of antibodies. Peroxisome proliferator
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receptor -co-activator-1 (PGC-1) was included as a primary metabolic regulator. The antibody
for Glut-1 did not provide adequate signal for analysis.
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Discussion
The principal objective of this study was to determine if a relatively mild or subthreshold
dose of hypoxia could suddenly trigger the HIF-1 cascade in heart. Promotion of HIF-1
protein accumulation and concomitant increases in expression for various target genes and their
proteins occurred within a time frame measured in minutes. To our knowledge, no prior study
has detailed the rapid triggering of the HIF-1 signaling pathway in heart by a relatively mild
hypoxic stress. Stroka et al showed organ variable response of HIF-1 to changes in ambient
oxygen in vivo over 1 hour (28). In their study, brain exhibited the greatest sensitivity with
marked induction at 18% FiO2, while kidney and liver showed no HIF-1 accumulation until
exposed to FiO2 6%. Although, those authors indicated that HIF-1 appeared in hypoxic heart,
no details regarding the experimental conditions were provided. In the current study we avoided
systemic induction of the HIF-1 pathway by employing an isolated perfused rabbit heart, which
showed minimal expression of this protein in the control group. Appearance of protein is
stimulated by the mild dose of hypoxia provided by the 10/110 sec short-cycle hypoxia protocol.
As HIF-1 mRNA was barely detectable in the control hearts, the array data imply that mild
short-cycle hypoxia increased mRNA content by promoting transcription rather than altering
mRNA stability. Rapid transcriptional regulation of this protein represents a novel mechanism
for heart, as changes in steady-state level have only been described as occurring after many hours
or even days (14).
HIF-1 mRNA appeared higher in the microarrays, although no HIF-1 protein
accumulated during the more severe hypoxic conditions (H/R 60/60), which also produced a
detectable drop in myocardial ATP concentration. Within the confines of this study performed in
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the intact heart, we are not certain whether lack of protein response reflects a transcriptional or
posttranslational disturbance. However, degradation of the HIF-1 proteins is triggered by
hydroxylation of two key proline residues in the O2 dependent degradation domain-containing
proteins via prolyl hydroxylase domain-containing (PHD) proteins. Most studies show
suppression of PHD activity by severe hypoxia, thereby inhibiting, but not suppressing HIF1degradation (3, 15). Therefore, our results suggest that inhibition of translation and not
accelerated protein degradation, contribute to the reduced HIF-1 protein accumulation in the
HR 60/60 group.
ATP depletion during severe hypoxia in HEK293 cells directly limits protein synthesis by
inhibiting activity of the mammalian target of rapamycin (mTOR) (5), which positively regulates
key enzyme controllers of translation initiation. We therefore considered if a relationship existed
between ATP and HIF-1 protein accumulation in the intact heart. Although, ATP depletion
occurs generally only during the most severe and often irreversible conditions of ischemia, the
same does not hold true during decreases in ambient O2 concentration, which are accompanied
by maintained or increased coronary flow rates. Accordingly, prior studies in heart in vivo (25,
26) and ex vivo (10) have shown that reducing oxygen concentration to just surpass the critical
threshold causes parallel depletion in phosphocreatine and ATP stores. While phosphocreatine
levels fully recover immediately with reoxygenation, ATP stores remain depressed due to
accelerated purine degradation and loss during hypoxia. Degradation and washout of the
degradation products is illustrated by the concomitant decrease in ATP and nondiffusable
nucleotides shown in the HR 60/60 (S) group, while maintaining the low levels of diffusible
nucleotides. Although, we did not measure phosphocreatine in this study, the stability of ATP in
the rabbit hearts subjected to the more modest HR 10/110 cycle implies that no major or
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sustained disruption in high energy phosphate metabolism occurred during the cycling or
afterwards. Additional support for limited disruption in energy status is the lack of increased
lactate production throughout the protocol in the HR 10/110 (A) group, implying that anaerobic
metabolism was not stimulated. Thus, the finding that HIF-1 accumulated after hypoxia with
stable ATP, but not under the more severe conditions accompanied by ATP depletion, indicates
that ATP either directly or indirectly enhances accumulation of this protein after hypoxia.
Rapid induction of HIF-1 target genes in heart by a reduction in O2 concentration has
not been previously examined in detail. Previously, Cai et al demonstrated that exposure of mice
to systemic intermittent hypoxia resulted in protection of isolated hearts against ischemiareperfusion 24 hours later. The mRNA for the HIF-1 target genes, VEGF, Glut1, and
erythropoietin (EPO) did not exhibit change after rather profound and prolonged systemic
hypoxia (five cycles of 6% O2 for 6 min alternating with 21% for 6 min), compared to the
hypoxic conditions in our current study. However, kidney EPO mRNA increased, and
presumably stimulated observed elevations in plasma EPO levels. Cardiac protection and EPO
elevation were abrogated in mice heterozygous for a knockout allele at the locus encoding HIF1 . Accordingly, those authors attributed cardiac protection primarily to hypoxic induction of
renal EPO mRNA.
Some of our mRNA results were obtained by pooling samples on microrarray chips in
order to identify potential targets for further evaluation, including immunoblot analyses of
respective proteins. We used strict criteria including two-fold differences in expression for
defining upregulation. Nevertheless, we cannot validate these array results through statistical
methods, even though they were for the most part consistent with Northern blot and protein
analyses. Our protocol using mild short-cycle hypoxia, free of systemic influence, produced
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protein elevations for multiple HIF-1 targets. Additionally we statistically validated elevation
for mRNA elevations for the putative HIF-1 target Grp94, as well as the stress related gene,
HSP70. Protein for the HIF-1 target gene, VEGF, rapidly accumulates, though levels are lower
in hearts exposed to the more severe protocol. Therefore, the data imply, that mild short-cycle
hypoxia rapidly initiates the HIF-1 signaling cascade through both transcriptional and
posttranscriptional modes. As expected, the transcriptional and posttranscriptional response
varies according to target gene. This variation may be related to target gene sensitivity or
differences in translational efficiency, which are uncovered by this short protocol. Additionally,
significant protein accumulation for HIF-1 and VEGF may be technically easier to detect, as
control levels for these are relatively low compared to the other proteins evaluated. The EPO
response to protocols, which presumably activate HIF-1 cascade, has varied (2, 14). Some
report that HIF-1 dramatically induces responses to prolonged heat stress (14). Our findings
with respect to EPO are consistent to results from previous studies employing longer and more
severe intermittent hypoxia (2). Accordingly, results from prior work and our own current
experiments taken together imply that hypoxia does not promote changes in cardiac EPO mRNA.
Although our study was directed towards evaluation of the HIF signaling cascade, the
commercial arrays also provide some interesting potential information regarding other important
pathways. We did not intend to pursue these serendipitous results within the scope of the current
study, although some of these results point to directions for further research. For instance, we
noted a marked decrease in mRNA for EEF1A1 in the severe hypoxia group. Unlike other
peptide elongation or initiation factors (13), the response of this particular eukaryotic elongation
factor to hypoxia has not been previously reported. Our finding with respect to mRNA
expression for this factor, though preliminary, represents a potential mechanism for reduction in
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protein synthesis after severe hypoxic insult. Additionally, we noted that severe hypoxia
decreased expression for survivin (BIRC5), a known inhibitor of apoptosis (1). Changes in
mRNA for EEF1A1 and survivin did not occur during the modest hypoxia, which elicited
response in the HIF-1 pathway.
Limitations The nature of the short-cycle hypoxia as performed in these protocols
precluded measurement of many parameters, which serve as energy sensors within
cardiomyocytes. Conceivably, undetected and transient alterations in cellular redox state may
trigger activation of HIF-1 signaling cascade. AMP-activated protein kinase (AMPK) serves as
an energy-sensing protein, which responds to transient metabolic stresses and modulates multiple
metabolic and gene signaling pathways (27). AMPK activity in particular can be triggered
rapidly by hypoxia in heart (6), although increases in activity for this enzyme can take as long as
30 minutes (7). The relationship between AMPK and HIF-1 has not been clearly established in
heart, though this enzyme displays a critical function for stimulating HIF-1 transcriptional
activity in cancer cells.
Conclusions In summary, we have shown that HIF-1 cascade responds to relatively modest
short-cycle hypoxia. We define this modest short-cycle hypoxia as subthreshold, as this degree of
oxygen deprivation does not elicit major changes in cardiac function or ATP stores. The
subthreshold definition is supported further by diminished stimulation for coronary
vasodilatation and anaerobic metabolism, apparent in hearts exposed to mild short-cycle hypoxia.
In contrast, slightly more severe short-cycle hypoxia or oxygen deprivation, which surpasses the
threshold, is accompanied by decreases in ATP and nucleoside pool, and marked elevations in
coronary blood flow and lactate production. These metabolic changes occur with reductions in
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21
the HIF-1 and VEGF protein accumulation, suggesting that protein synthesis is inhibited,
although an increase in protein degradation remains a possibility. Induction of HIF-1 by
subthreshold hypoxia shows promise as a mechanism for cross-adaptation and protection against
subsequent ischemic injury. Thus, mild short-cycle hypoxia shows clinical relevance or
potential, as this protocol stimulates this signaling cascade within a brief time without causing
detectable changes in cardiac function or energy status. The data also highlight the exquisite
sensitivity of the HIF pathway to subthreshold oxygen deprivation.
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Acknowledgements
This work was supported in part by the grants from Children’s Hospital and Regional Medical
Center (HR5836), NSFC 38970307, and NIH (R01HL60666).
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25
Figure legends
Figure 1. Phasic recording of left ventricular pressure (LVP) and coronary flow (CF) during
control and short-cycle hypoxia. A. Phasic tracings are stable for 90 min in a control heart
without hypoxic exposure. B. Tracings are from baseline and cycle 23 using a 30 second/90
second hypoxia and reoxygenation protocol. O2 content for the hypoxia cycle was 2.2 µl/ml.
LVP is already diminished from baseline at the start of the 23rd cycle. During the subsequent
hypoxia portion of the cycle, LVP decreases further as CF increases. Some recovery occurs to
levels less than baseline during reoxygenation.
Figure 2. Hypoxic intensity injury threshold for cardiac functional recovery. Perfusate O2
content is plotted against percent left ventricular developed pressure (DP). Short-cycle hypoxia
was performed using hypoxia/reoxygenation cycling of 10 sec/110 sec and continued for 23
cycles. DP is shown for the final reoxygenation phase. Each point represents the mean ± SE for
4-6 hearts exposed to a single hypoxia intensity dose. O2 content below 2.2 Kl/ml impairs DP
recovery.
Figure 3. Injury threshold determined by cycle length and number. Maximal left ventricular
developed pressure (DP) during the reoxygenation phase is plotted per cycle. Short-cycle hypoxia
was performed with O2 content of 2.2 Kl/ml. Hypoxia/reoxygenation (H/R) cycles are 10sec/110
sec (n=4), 30sec/90 sec (n=4), and 60sec/60 sec (n=8). The abbreviations used are: C = Control
(n=8). DP recovery does not differ significantly between control and the H/R=10/110 sec
groups.
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26
Figure 4. Coronary flow responses. Coronary flow (CF) is plotted for the hypoxic portion of the
first (H1) and twenty-third cycles (H23) as a percentage of baseline. The abbreviations further
defined in the text are: A, hearts exposed to hypoxia/reoxygenation (H/R) cycling protocol at
10sec/110sec; C, Control, no hypoxia cycling; S, severe hypoxia, using H/R=60sec/60sec. N = 4
per group.
Figure 5. Relationship between the injury threshold and energy status. The abbreviations further
defined in the text are: A,hearts exposed to hypoxia/reoxygenation (H/R) cycling protocol at
10sec/110sec; C, Control, no hypoxia cycling; S, severe hypoxia, using H/R=60sec/60sec; TDN=
Total diffusible nucleosides; TNN=total non-diffusible nucleotides. n = 4 in each group; *,
P<0.05 vs. C.
Figure 6. Lactate production for the three groups. Data is shown for the first short-cycle
hypoxia portion (H1) and the final cycle, H23. A, hearts exposed to hypoxia/reoxygenation
(H/R) cycling protocol at 10sec/110sec; C, Control, no hypoxia cycling; S, severe hypoxia, using
H/R=60sec/60sec; *, P<0.05 vs. C, n = 4 per group.
Figure 7. Changes in mRNA levels for HSP70-1 and Grp94. The upper panels are the
representative Northern blot for HSP70-1 (right) and Grp94 (left). Each lane was loaded with 15
µg total RNA from ventricular myocardium and probed specifically for HSP70-1, Grp94 and
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The lower panel shows steady-state
mRNA levels that are normalized to GAPDH and relative to the hearts in control (C). A, hearts
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27
exposed to hypoxia/reoxygenation (H/R) cycling protocol at 10sec/110sec; C, Control, no
hypoxia cycling; S, severe hypoxia, using H/R=60sec/60sec, n = 4 per group.
Figure 8. Hypoxia microarray. The figure shows the differences in gene expression between the
groups A, C, S. Abbreviations are: ADM= Adrenomedullin; BIRC5= Apoptosis inhibitor 4
(Survivin); COL1A1= Collagen, type 1, alpha 1; GAPD= Glyceraldehyde-3-phosphate
dehydrogenase; Glut1= Solute carrier family number1; HIF1 = Hypoxia inducible factor-1 ;
IL6ST=Interleukin 6 signal transducer; VEGF= Vascular endothelial growth factor. A,hearts
exposed to hypoxia/reoxygenation (H/R) cycling protocol at 10sec/110sec; C, Control, no
hypoxia cycling; S, severe hypoxia, using H/R=60sec/60sec.
Figure 9. Immunoblots for HIF-1 and VEGF. The upper panels show protein levels that are
normalized to -actin and relative to the hearts in control (C). The lower panels show
representative Western blots for HIF-1 (left) and VEGF (right) proteins. A,hearts exposed to
hypoxia/reoxygenation (H/R) cycling protocol at 10sec/110sec; C, Control, no hypoxia cycling;
S, severe hypoxia, using H/R=60sec/60sec; *, P<0.05 vs. C, n = 4 in each group.
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Table 1.
28
Hemodynamics (Mean±SE)
Baseline
H1
R1
H23
R23
REO 45 min
EDP (mmHg)
C
S
1.8±0.2
2.7±0.5
1.9±0.3
5.1±1.0*
2.1±0.2
4.8±1.1
3.2±0.9
8.1±3.0*†
2.8±0.6
5.2±1.2*†
3.6±1.4
6.7±3.2
PSP (mmHg)
C
S
134.5±1.4
130.7±5.3
134.2±2.1
52.8±14.1*†
135.5±2.0
130.9±2.0
135.8±2.5
47.6±4.4*†
134.3±2.6
114.9±3.1*†
126.9±3.4
110.6±5.8*†
DP (mmHg)
C
S
132.6±1.6
128.0±5.5
132.3±2.2
47.7±13.9*†
133.4±1.9
126.1±3.0
132.6±2.8
39.5±2.2*†
131.5±2.8
109.7±4.2*†
123.3±4.5
103.9±4.8*†
dP/dtmax (mmHg/sec)
C
2185±101
S
2059±99
2176±107
869±64*†
2201±99
1864±81
2165±112
618±50*†
2167±120
1589±173*
1970±151
1573±131
-dP/dtmax (mmHg/sec)
C
1638±68
S
1527±72
1656±65
736±71*†
1647±64 1
1519±57
581±70
521±16*†
1615±77
1282±123
1475±88
1227±75
HR (Beats/min)
C
206.5±10.4
S
191.3±15.8
206.3±10.6
190.0±30.3
208.3±9.8
188.5±16.3
209.5±10.7
175.0±24.5
208.8±11.0
175.0±27.3
200.8±10.1
183.3±22.0
PRP (103 mmHg/min)
C
27.39±1.39
S
24.32±1.55
27.30±1.55
7.92±2.23*†
27.78±1.35
23.66±1.59
27.85±1.91
6.77±0.69*†
27.53±1.97
19.26±3.08
24.80±1.80
18.88±1.91
CF (ml/min/g)
C
11.5±0.37
S
10.6±1.08
11.6±0.15
16.7±0.58*†
11.2±0.37
11.8±0.21
12.0±0.60
17.0±1.20*†
12.0±0.61
12.8±0.90
10.7±0.82
7.9±0.92
MVO2 (Gmol/min/g)
C
6.42±0.13
S
5.71±0.53
6.07±0.21
0.59±0.10*†
6.14±0.43
6.15±0.17
6.36±0.36
0.53±0.12*†
6.51±0.25
5.79±0.49
6.19±0.61
4.60±0.61
The hemodynamic indexes were determined in isolated perfused hearts at baseline, short-cycle hypoxia, and
reoxygenation as described in Methods. The abbreviations used are: C, control group (n=4); S, protocol for
hypoxia/reoxygenation (H/R)=60sec/60sec, total of 23 cycles (n=4); CF, coronary flow; DP, developed pressure;
±dP/dtmax, maximum of the positive or negative first derivative of left ventricular pressure; EDP, end diastolic pressure;
HR, heart rate; PRP, product of HR and DP; PSP, the peak of left ventricular pressure; MVO2, myocardial oxygen
consumption. Hn or Rn refers to cycle portion, hypoxia or reoxygenation, and n=cycle number. REO 45 min refers to 45
min after completion of H/R cycling.*, P< 0.05, compared with C. †, P< 0.05, compared with baseline.
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Table 2.
Pathway
Steady-state mRNA response to short-cycle hypoxia
Gene
Hypoxia –inducible factor 1 (HIF-1 )
A/C
2.1
S/C
1.7
Adrenomedullin (ADM)
Erythropoietin (EPO)
Glucose regulated protein 94 (Grp94)*
Heme oxygenase 1 (HEME1)
Insulin-like growth factor 2 (IGF-II)
Nitric oxide synthase 2A (NOS)
Solute carrier family number1
(SLC2A1, GLUT1)
Solute carrier family number4
(SLC2A4, GLUT4)
Vascular endothelial growth factor
(VEGF)
1.0
1.0
1.5
2.0
3.1
1.6
1.0
0.6
3.3
2.6
1.4
1.2
11.4
8.2
2.6
3.3
1.6
5.3
0.95
1.21
1.7
1.8
0.55
0.61
2.0
4.8
0.96
2.2
2.7
4.0
4.2
4.0
2.0
3.5
19.5
0.34
2.5
3.8
3.8
8.3
3.8
2.5
3.0
36.5
8.7
0.84
5.2
3.3
13.6
0.51
15.5
6.9
HIF1 target genes
Other Hypoxia/Ischemia related genes
Apoptosis inhibitor 4 (Survivin)
Collagen, type 1, alpha 1 (COL1A1)
Connective tissue growth factor (CTGF)
Endothelin converting enzyme 1 (ECE1)
Eukaryotic translation elongation factor 1
alpha 1 (EEF1A1)
Epidermal growth factor receptor (EGFR)
Formin binding protein 3(HYPA)
Glucose phosphate isomerase (GPI)
Heat shock protein 70-1 (HSP70-1)*
Interleukin 6 signal transducer (GP130)
Leukocyte receptor cluster member (BB1)
Plasminogen activator, urokinase (uPA)
Proteasome subunit, beta type, 3 (PSMB3)
Protein phosphatase 2, catalytic subunit,
beta isoform (PPP2CB)
Retinoic acid receptor, alpha (RARA)
Ribosomal protein S2 (RPS2)
Ribosomal protein L37 (RPL37)
Data reported as ratios among groups are derived from microarray chip intensities
normalized by the average value of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and -actin in the same membrane. The abbreviations for groups A, C, and
S are defined in the text. We defined upregulation by array compared to control as
ratio greater or equal to 2.0, and downregulation as ratio less than or equal to 0.6. *
indicates analyses by Northern blot, and P < 0.05 (n = 4), as noted in Figure 7.
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Table 3. Protein contents response to short-cycle hypoxia
Protein
Glucose Transporter 4
Glucose regulated protein 94
Interleukin 6 signal transducer
Heat shock protein 70
Heme oxygenase 1
Peroxisomal proliferator gamma
coactivator-1
Vascular endothelial growth factor
Hypoxia inducible factor 1
A/C
S/C
(Glut4)
(Grp94)
(IL6ST)
(HSP70)
(HMOX1)
0.97±0.06
0.77±0.08
0.55±0.14
0.82±0.32
1.06±0.11
0.90±0.09
0.74±0.10
0.67±0.15
0.84±0.31
1.21±0.12
(PGC1)
(VEGF)
(HIF1 )
0.88±0.09
5.57±1.06*
5.27±1.14*
1.15±0.12
2.86±0.98
0.50±0.11
The abbreviations used are: A, A group (10sec/110sec) as described in the text; C=,
Normal control group; S, S group (60sec/60sec); A/C, the ratio of A group vs. normal
control; S/C, the ratio of S group vs. control. * = P<0.05.
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* *
CF (% of Baseline)
180
160
*, P<0.05, vs. C
140
120
100
H1
H23
80
60
40
20
0
C
A
S
Ning-MOLRESP-Fig 4.eps
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30
*=P<0.05 vs. C
µmol/g dw
25
*
20
*
C
A
S
15
10
5
0
ATP
TNN
TDN
Ning-MOLRESP-Fig 5.doc
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(µmol/min/g ww tissue)
LACTATE
25
*=P<0.05 vs. C
*
20
15
*
BS
H1
H23
10
5
0
C
A
S
Ning-MOLRESP-Fig 6.doc
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Ning-MOLRESP-Fig 7.doc
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Ning-MOLRESP-Fig 8.doc
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Ning-MOLRESP-Fig 9.doc
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