| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Divisions of Cardiology and Genetics and Development, Department of Pediatrics, University of Washington, Seattle 98195; and Children's Hospital and Regional Medical Center, Seattle, Washington 98105
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Hypothermia improves resistance to
ischemia in the cardioplegia-arrested heart. This adaptive
process produces changes in specific signaling pathways for
mitochondrial proteins and heat-shock response. To further test for
hypothermic modulation of other signaling pathways such as
apoptosis, we used various molecular techniques, including cDNA
arrays. Isolated rabbit hearts were perfused and exposed to
ischemic cardioplegic arrest for 2 h at 34°C
[ischemic group (I); n = 13] or at 30°C
before and during ischemia [hypothermic group (H);
n = 12]. Developed pressure, the maximum first
derivative of left ventricular pressure, oxygen consumption, and
pressure-rate product (P < 0.05) recovery were superior in H compared with in I during reperfusion. mRNA expression for the mitochondrial proteins, adenine translocase and the 
-subunit of F1-ATPase, was preserved by hypothermia. cDNA
arrays revealed that ischemia altered expression of 13 genes.
Hypothermia modified this response to ischemia for eight genes,
six related to apoptosis. A marked, near fivefold increase in
transformation-related protein 53 in I was virtually abrogated in H. Hypothermia also increased expression for the anti-apoptotic Bcl-2
homologue Bcl-x relative to I but decreased expression for
the proapoptotic Bcl-2 homologue bak. These data
imply that hypothermia modifies signaling pathways for
apoptosis and suggest possible mechanisms for
hypothermia-induced myocardial protection.
-subunit of F1-ATPase; hypothermic
adaptation; myocardial reperfusion
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
MODERATE HYPOTHERMIA IMPROVES resistance to oxidative injury in the heart. Hypothermic protection can be proffered by application of cold during ischemic insult. In addition, cold stress applied before warm ischemia initiates adaptive responses and promotes preservation of cardiac contractile function (24). This cross-adaptive phenomenon might represent thermotolerance transfer. Hypothermic adaptation is also linked to reduced ATP store depletion during ischemia and reperfusion (27, 28) and is further characterized by enhanced postischemic gene expression for specific stress-related proteins and constitutive mitochondrial membrane proteins (28, 29).
Although modulation of energy metabolism contributes to protection afforded by hypothermia, other injury-reducing mechanisms might also operate in response to cold. Elucidation of these mechanisms could lead to identification of specific molecules that provide protection during and after ischemic insult. Recent experiments in noncardiac tissues indicate that cold modifies various cell signaling pathways. Specifically, investigations performed with cultured neuronal tissues, or in brain in vivo, indicate that mild or moderate hypothermia mitigates apoptosis, programmed cell death induced by hypoxia or ischemia (5, 22, 31). In addition, several studies demonstrate that cardiomyocyte apoptosis contributes to ischemia-reperfusion injury (3, 4, 10, 13, 32). Adaptive processes such as ischemic preconditioning are found to ameliorate reperfusion injury partially through modifications of specific cell death signaling pathways (21). These findings formed the basis for a hypothesis stating that hypothermia similarly induces myocardial protection by affecting the signaling for apoptosis and/or other pathways after ischemia and reperfusion. Testing this hypothesis was the principal objective of this study.
The availability of cDNA arrays provides an objective means to examine the molecular profile for multiple genes and identification of changes in signaling pathways in a heart altered by ischemia and hypothermia. This technique and the confirmatory methods, including Northern blot and reverse transcriptive-polymerase chain reaction (RT-PCR), enable testing of the principal hypothesis. Changes in expression of specific genes were associated with functional improvement in cardiac function induced by hypothermia.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Preparation of isolated heart. Thirty rabbits (male or female, 2.3-2.8 kg body wt) were anesthetized with pentobarbital sodium (45 mg/kg iv) and heparinized (700 U/kg iv). Each heart was rapidly excised and immersed momentarily in ice-cold physiological salt solution, pH 7.4, containing (in mmol/l) 118.0 NaCl, 4.0 KCl, 22.3 NaHCO3, 11.1 glucose, 0.66 KH2PO4, 1.23 MgCl2, and 2.38 CaCl2. The aorta was cannulated in the Langendorff mode, and the heart was perfused with physiological salt solution that had been equilibrated with 95% O2-5% CO2 at 37°C and passed twice through filters with 3.0-µm pore size. Perfusion pressure was maintained at 90 mmHg (23). An incision was made in the left atrium, and a fluid-filled latex balloon was passed through the mitral orifice and placed in the left ventricle. The fluid from the Thebesian vein did not accumulate in this preparation. The balloon was connected to a pressure transducer for continuous measurement of left ventricular pressure (LVP) and measurement of the first derivative with respect to time. The caudal vena cavum, the left and right cranial vena cava, and the azygous vein were ligated. The pulmonary artery was cannulated to enable collection of coronary flow, measured with a flow meter (T201, Transonic Systems, Ithaca, NY).
Analog signals were continuously recorded on an online computer (Macintosh, Biopac analog signal acquisition system) and a pressurized ink chart recorder (Gould, Cleveland, OH). To characterize cardiac function, we defined developed pressure (DP) as peak systolic pressure minus end-diastolic pressure. Calculating the product of heart rate and DP (PRP) provided an estimate of myocardial work (mmHg/min). Myocardial oxygen consumption (M
O2) was calculated as MO2 = CF × [(PaO2 
PvO2) × (c/760)], where CF is the coronary flow
(ml · min
1 · g wet
tissue1), PaO2 PvO2 is the difference in the
PO2 (Torr) between perfusate (arterial) and
coronary effluent (venous), and c is the Bunsen solubility
coefficient of O2 in perfusate at 37°C (22.7 µl
O2 × atm1 × ml1)
(26, 28). Oxygen extraction was calculated as
MO2 divided by oxygen content in the
perfusate. The wet weight of the heart was determined at the conclusion
of each experiment after the fat and great vessels were trimmed and
then the heart being blot dried with nine-layer cotton gauze.
Procedures followed were in accordance with institutional and National
Institutes of Health guidelines.
Lactate, pH, and CO2 measurements.
The coronary inflow and effluent were collected; concentrations of
O2 and CO2 were immediately measured with a
Radiometer (ABL 5, Copenhagen, Denmark). The difference in
CO2 content between the coronary effluent and inflow was
calculated as (PvCO2 PaCO2) × c/Vm, where
PvCO2 PaCO2 is the difference
in the PCO2 (Torr) between coronary effluent
(venous) and perfusate (arterial), c is the solubility
coefficient of CO2 in perfusate at 37°C (0.53 ml
CO2 × atm1 × ml1
perfusate), and Vm is the molar volume (22.4 × mM1) (26, 28). Lactate concentration
was measured with a GM7 analyzer (Analox Micro-Stat, London, UK).
RNA isolation.
After removal of excess fat and connective tissues, the left
ventricular wall was briefly blotted on nine-layer gauze, frozen in
liquid nitrogen, and then stored at 80°C. An aliquot of the frozen
heart tissue (100-200 mg) was pulverized and homogenized, and the
total RNA was then extracted with a RNA isolation kit (Ambion, Austin,
TX). RNA samples were tested by ultraviolet absorption at 260 nm to determine the concentration. The quality and
concentration of the RNA samples were further confirmed by
electrophoresis on denatured 1% agarose gels (28, 30).
cDNA arrays. The array filters were obtained from Clontech Laboratories (Palo Alto, CA) and Super Array (Bethesda, MD). The Clontech filters had 234 known gene fragments, representing a wide range of genome functions. The Super Array filters contained 68 cDNA gene fragments, immobilized and duplicated on a nylon membrane with specific pathway functions, including transcription signals, stress-inducible apoptosis, DNA repair functions, and mitogenic signal pathways. Procedure was followed as recommended by the companies with minor modifications.
For each experiment, 20 µg of total RNA or 1 µg of poly(A) RNA was used for first-strand synthesized cDNA (SuperScript first-strand synthesis system for RT-PCR, GIBCO BRL, Life Technologies, Gaithersburg, MD). The RNA sample was mixed with 2 µl of 10× cDNA synthesis primer mix and then added to distilled H2O to a final volume of 6 µl. Incubated tubes were placed at 70°C for 2 min to reduce the temperature of the thermal cycler to 50°C. Fourteen microliters of "master mix" were added, which contained 2× dNTP, 5 mM DTT, [
-32P]dATP
(3,000 Ci/mmol), and 50 units of Moloney murine leukemia virus reverse
transcriptase. Tubes were incubated at 50°C for 25 min. The reaction
was then stopped by adding 1 µl of 10× termination mix. The
[32P]cDNA was purified from an unincorporated
[32P]dATP by a column chromatography method. The labeled
cDNA probe was hybridized overnight in each filter at ~1 × 10 6 counts · min1 · ml1. After
filters were washed, they were exposed on a PhosphorImager (model 400S,
Molecular Dynamics, Sunnyvale, CA) and on Kodak BioMax film (Eastman
Kodak, Rochester, NY) at 70°C for various amounts of time.
Specific array signal spots were analyzed with ImageQuant
quantitation software (Molecular Dynamics). The relative levels of cDNA
expression were assessed by comparing the same signals obtained in
normal control, ischemic, and hypothermic conditions. Equivalent amounts of total RNA from five individual and randomly selected heart samples within each experimental group were combined to
form subgroups. Instead of testing according to individual sample, the
samples were tested according to subgroup to diminish systemic error.
The subgroup samples were pooled on each array and repeated four to
five times in different membranes. The membranes were stripped and
reused two to three times. Relevant changes in gene expression were
identified by using decision tree classification (6) and
linear discriminant analysis (2). These methods have been
adapted for use in arrays by the Fred Hutchison Cancer Research Institute.
RT-PCR analysis.
Five micrograms of total RNA were added in 20 µl of reaction mixture
and preheated for 10 min at 65°C with 100 ng of
poly(dT)12-18 primer. The first-strand cDNA was
synthesized by SuperScript II RNase H-RT at 42°C for 50 min (Life
Technologies). The reaction was terminated at 70°C after 15 min. For
subsequent PCR reaction (50 µl), 1 µl of the cDNA mixture was used
for each gene-specific amplification. The gene-specific primers for p53
and -actin were designed by a primer design program (Primer Design
3, Scientific and Educational Software, State Line, PA).
-Actin primers were as follows:
5'-CGAGCGGGAAATCGTGCGTGACATTAAGGAGA-3' (-actin478
forward), 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'
(-actin478 reversed),
5'-AAAGACCTGTACGCCAACACAGTGCTGTCTGG-3' (-actin229 forward), and 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'
(-actin229 reversed).
Amplification reactions were conducted in 25-50 µl of reagent
mixture with an initial step of 94°C for 3 min followed by 25-35 cycles of amplification, depending on cDNA abundance in preparations. Each cycle was at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and finally at 72°C for 7 min. The -actin was used as a reference control.
PCR products were analyzed by agarose gel electrophoresis. PCR bands of
the predicted size were isolated and subjected to sequencing (ABI Prism
310, Perkin-Elmer Foster) to confirm correct gene identities.
Expression levels of specific genes were quantified by Image Station
440cf (Eastman Kodak) on ethidium-stained bands of amplified fragments
after normalization to -actin.
Northern blot analysis.
For Northern blot analysis, 15 µg of RNA were denatured and
electrophoresed in a 1% formaldehyde agarose gel, transferred to a
nitrocellulose transfer membrane (Micron Separations, Westboro, MA),
and cross-linked to the membrane with a short-wave ultraviolet cross-linker. The prehybridizing and hybridizing solutions contained 50% formamide, 1× Denhardt's solution, 6× SSPE, and 1% SDS. cDNA probes were labeled with [32P]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 h. The blots were then washed several times
with a final wash in 1× standard sodium citrate and 0.1% SDS at
65°C. The blots were exposed through a PhosphorImager (model 400S)
and/or on Eastman Kodak BioMax film at 70°C. The relative amounts
of mRNA were measured by using ImageQuant quantitation software
(Molecular Dynamics). The same size area at each band was taken to
measure the intensity, and the same size area at the closest upstream
position of each band was used as the background of the image. RNA
loading was normalized by comparison to that of 28S and/or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (28, 30).
mRNA levels of the -subunit of F1-ATPase
(F1-ATPase) were detected by using a 1.8-kb cDNA fragment cloned from the human HeLa cell line (American Type Culture Collection, Manassas, VA) (28, 30). Adenine nucleotide
translocator isoform 1 (ANT1) mRNA levels were detected by
using a 1.4-kb cDNA fragment cloned from human skeletal muscle
(American Type Culture Collection) (28, 30). To compare
different mRNA levels in the same myocardial sample, 15-µg aliquots
of total RNA from the myocardium were analyzed by means of sequentially
reprobing the membranes with GAPDH carriers (Clontech),
F1-ATPase, and ANT1 cDNA probes.
Experimental protocols. The experimental model used in these experiments has been previously described in detail (26, 28). After we completed instrumentation and performed calibrations, left ventricular balloon volumes were varied over a range of values to construct left ventricular function curves. In this manner, it is possible to define a specific balloon volume that is associated with a DP between 100 and 140 mmHg. This volume remained unchanged during baseline, ischemia, and reperfusion conditions. The intraventricular balloon volumes were not adjusted to produce specific end-diastolic pressures; rather, we defined a level of systolic pressure development. However, end-diastolic pressures at baseline >10 mmHg were not accepted (23). Excluded were data from hearts with DPs <100 mmHg or >140 mmHg. Baseline data were obtained after a 30-min equilibration period. The same procedures were followed in each experiment. During the baseline period, data were obtained with the hearts maintained at 37°C with a temperature-controlled organ bath.
Figure 1 summarizes the hypothermia protocol. To adjust the infused temperature, the myocardial and pulmonary outflow temperatures were monitored continuously with thermal probes. The rabbits were divided into an ischemic group (at 34°C ischemia, n = 13) and a hypothermic group (at 30°C, n = 12). The hypothermic group was treated with hypothermia before and during ischemia. Hemodynamic data were recorded for 45 min during reperfusion, followed by immersion of the tissue in liquid nitrogen for the Northern blot analysis.
|
Statistical analysis. The values are reported as mean ± SE. The Statview 4.5 (FPV) program (Abacus Concepts, Berkeley, CA) was used for statistical analysis. Data were evaluated with repeated-measures ANOVA within groups and with single-factor ANOVA across groups. When significant data were obtained with Scheffé's F test, individual group means were tested for differences with F values (two-tailed test). The criterion for significance was P < 0.05 for all comparisons (30).
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Functional recovery during reperfusion.
The heart weight and left ventricular balloon volume were similar in
the ischemic (6.80 ± 0.45 g and 1.60 ± 0.045 ml, respectively) and the hypothermic treatment groups (6.15 ± 0.25 g and 1.46 ± 0.034 ml, respectively). Under the
baseline conditions, there appeared to be no significant difference
between the groups in end-diastolic pressure, DP, maximum of the
positive or negative first derivative of LVP with respect to time,
heart rate, PRP, and MO2. Hemodynamic
results are summarized in Table 1 and Fig. 2. The hypothermia-treated hearts
demonstrated superior functional recovery compared with the
ischemic group.
|
|
Ischemic contracture. As noted in MATERIALS AND METHODS, we used a specific balloon volume that was adjusted and maintained throughout the protocol. This allowed comparisons of LVP under constant end-diastolic volume. After we injected the cardioplegic solution, the LVP was always near 0 mmHg. We defined the beginning of ischemic contracture by the initial rise in LVP above 2 mmHg. Ischemic contracture began in control hearts after 69.8 ± 4.4 min of ischemia. No ischemic contracture was observed after 120 min of ischemia in the hypothermia-treated hearts.
Accumulation of catabolic products.
An obvious increase in accumulation of lactate and CO2 was
noted in ischemic hearts at 120 min of ischemia (Fig.
3). Accumulation of both metabolites was
significantly lower in hypothermic hearts. These data imply that
aerobic and anaerobic metabolic rates were markedly decreased with
hypothermia treatment.
|
Gene expression for mitochondrial-specific proteins.
Northern blot analysis showed that F1-ATPase and
ANT1 mRNA were preserved by hypothermic treatment. The
intensity of F1-ATPase and ANT1 mRNA were
normalized to GAPDH. mRNA F1-ATPase levels were 3.0 ± 0.4 greater in the hypothermic than in the ischemic group
(P < 0.01). mRNA ANT1 levels were 2.9 ± 0.3 greater in the hypothermic than in the ischemic group
(P < 0.01) (Fig. 4).
|
cDNA array analysis.
cDNA arrays were used to determine the abundance of expressed genes in
hearts from the normal control, ischemic, and
hypothermic-ischemic groups (Fig. 1). The arrays consisted of
the following functional gene groups: 1) stress response
regulator and effector; 2) apoptosis, DNA synthesis,
damage repair, and replication; 3) drug metabolism; 4) mitogenic signal pathways; 5) other signal
pathways; 6) transcription factors and DNA binding proteins;
and 7) receptors, cell-surface antigens, and adhesions.
cDNAs of -actin and GAPDH served as controls.
), cyclin-dependent kinase inhibitor 1A
(p21waf1), and transformation-related p53, as
well as others. Seven of the 10 genes belonged to the apoptosis
pathways (p53 signal pathway). Hypothermic treatment decreased the
hybridization signal in 5 of the 10 genes. There was a 4.7-fold
increase in p53 expression in the ischemic group compared with
the control group; hypothermia reversed it to 0.15-fold of that shown
for the ischemic group (Table 2, Fig.
5). The hybridization signal increased
for three genes during hypothermic treatment, including a gene of
anti-apoptotic Bcl-2 homologue (Bcl-x), whereas the
proapoptotic Bcl-2 homologue, bak, exhibited a
significant decrease in expression.
|
|
RT-PCR analysis.
Changes in p53 expression were further confirmed by RT-PCR
amplifications of the control, ischemic, and hypothermic groups (Fig. 6). Correct identification of the
p53 sequence was first verified by DNA sequencing (results not shown).
The relative intensity of p53 expression in the ischemic group
compared with normal was greater than fourfold. Expression in the
hypothermic group displayed expression 0.3 times that of the
ischemic group (P < 0.05). Thus the RT-PCR
results correspond to the findings of cDNA arrays.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Hypothermia ameliorates oxidative injury induced during myocardial ischemia and reperfusion. Accordingly, cardiac function during reperfusion in this study was improved by application of hypothermia beginning before and extending through the ischemic period. Hypothermic protection is generally attributed to elevations in the energy supply-to-demand ratio during ischemia. This has been previously substantiated in ischemic cardioplegia-arrested rabbit heart by superior ATP preservation induced by hypothermia applied either before (28) or during ischemia (29). High-energy phosphate store preservation is subject to a critical temperature threshold (30°C), above which this advantage rapidly dissipates (27). The current experiments employed hypothermic temperatures both before and during ischemia at 30°C. This protocol thus provides adaptation, characteristic of cold tolerance, and resistance to injury during the oxidative insult. Complementing these two protective mechanisms of hypothermia optimizes metabolic, functional, and gene expression differences between this experimental group and the ischemic group. Elevations in lactate and CO2 production throughout ischemia occurred in the normothermic group, thus confirming increased energy utilization compared with the hypothermic hearts throughout the ischemic period (27, 28).
Previous studies in this laboratory indicate that hypothermia applied
either before or during ischemia promotes gene expression for
the constitutive mitochondrial membrane proteins: ANT and F1-ATPase. This phenomenon, confirmed in the present
study, occurs along with accelerated induction of gene expression for
heat shock protein 70 (26, 28, 29) and emulates
the induction of these genes by cold stress in cold-adapted tissues
(14). The previous investigations addressed expression for
these specific genes and did not evaluate other pathways that could be
altered by hypothermia. Subsequently, several investigators noted
participation of the ANT in formation of the permeability transition
pore complex (PTPC) within the inner mitochondrial membrane. This
complex presumably promotes leakage of protons and ions from
the mitochondrial matrix and leads to loss of mitochondrial integrity,
initiating sequence activation of apoptotic pathways. ANT protein
expression tightly coordinates with steady-state mRNA levels in several
animal models. Thus one might presume that the elevated ANT gene
expression in the hypothermic hearts after reperfusion heralds
accelerated ANT synthesis and accumulation in the mitochondrial
membrane. This process could lend stability to the mitochondrial
membrane and thwart mechanisms leading to apoptosis, now a
recognized mechanism of cardiomyocyte death following oxidative injury
(3, 4, 10, 11, 13, 19). These speculations led to the
consideration that hypothermia could alter other pathways related to
mitochondrial membrane integrity and stability.
Expression patterns for various genes documented in this study support the validity of this hypothesis. A principal finding in this study was that a relatively mild degree of hypothermia modified postischemic gene expression for several proteins that contribute to the regulation of apoptosis. Hypothermia blunted postischemic expression for the transformation-related p53 (tumor suppressor p53) as well as for various genes that are considered its targets. Previous studies have linked p53 protein expression with the morphological changes and genomic DNA fragmentation characteristic of apoptosis after oxidative stress in cardiomyocytes (19). The p53 protein serves as a transcriptional activator of a number of target genes, including Gadd45 (8, 37), p21, caspase-3, and others (33). The temperature-induced modifications in p53 response to ischemia in heart have not been previously noted and represent an area for further research.
Changes in expression for two members of the Gadd45 family
during ischemia represent the type of serendipitous results
often yielded by cDNA microarray analyses. Hypothermic modification of
the Gadd45 and Gadd45 responses to
ischemia represents an additional and novel finding generated
by cDNA array technique. The Gadd45 family is expressed in a
variety of tissues and is upregulated by hypoxia or stress (10,
13, 33). An extensive literature review yielded only a single
previous evaluation of expression for this gene in the heart
(34). Specifically, Rees (34) noted
Gadd45 expression decreases in rat heart during fetal development. p53 upregulates Gadd45, which inhibits DNA
synthesis and allows repair of damaged DNA in specific cell types
demonstrating mitotic activity. However, the functions of
Gadd45 and Gadd45 have not been examined in
cardiomyocytes. Therefore, the implications of changes in expression
for the Gadd45 genes in heart are undetermined.
The increased expression of the p21 gene after ischemia is consistent with the hypoxic response in cardiomyocytes previously demonstrated by Long and coauthors (19). Although p21 is a target gene of p53 (8), its expression can also be induced independently by reactive oxygen species (35). Therefore, lack of suppression of the p21 response by hypothermia might reflect induction by a separate pathway, not regulated by p53 or subject to thermoregulation. In addition, failure of hypothermia to modify response of p21 expression to ischemia, as well as that of egr-1, demonstrates that hypothermia modification is not ubiquitous and might indicate the presence of specific thermally activated cofactors.
Mitochondrial membrane integrity depends on Bcl-2 family expression. Members of the Bcl-2 gene family both positively and negatively regulate apoptosis. Bcl-2 localizes in the cytoplasmic face of the mitochondrial outer membrane, endoplasmic reticulum, and nuclear envelope (1, 16). The Bcl-2 protein prevents the cascade leading from the opening of the PTPC complex to cytochrome c release, caspase activation, and cell death. Regulation of apoptosis is highly dependent on the ratio of antiapoptotic to proapoptotic proteins. The proapoptotic Bcl-2 family member bak, considered in the present study, alters mitochondrial stability and enhances apoptotic cell death (17), whereas Bcl-xl forms antiapoptotic heterodimers with Bcl-2. Our results confirm findings from previous studies by demonstrating that bak and Bcl-x are not highly expressed in normal heart tissue (7, 20). However, hypothermia markedly accentuates ischemic induction of Bcl-x and abrogates bak expression. Thus the hypothermia application modifies Bcl-2 family expression in directions that would be expected to stabilize the mitochondrial membrane and prevent apoptosis.
The majority of microarray studies have not reported an analysis of specificity (18). Nor have strategies for identification of differences in array signal intensities been well described. This lack of knowledge could diminish the value of the current data and represent a limitation of this study. However, a recently published analysis of arrays in heart demonstrated that specificity can be increased substantially by performing repetitive experiments. We repeated array experiments at least four to five times to confirm reproducibility of our results. In addition, traditional methods of discrimination were modified to analyze these arrays and limit false-positive results. Finally, RT-PCR confirmed changes in gene expression for p53. RT-PCR results for this gene were virtually identical to results obtained from array analysis.
In summary, through use of cDNA arrays, these experiments identified
the induction of several genes by ischemia in heart. In
particular, hypothermic modification of p53 and ischemic
induction of Gadd45 and Gadd45 are novel
findings in cardiac tissue. Furthermore, hypothermic modification of
several signaling pathways in directions presumed to promote
antiapoptotic factors was documented. These findings, paired with
preserved gene expression for constitutive mitochondrial proteins imply
that hypothermia promotes signaling for mitochondrial membrane
stability after ischemia-reperfusion injury.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. W. G. Guntheroth, Department of Pediatrics, for advice.
| |
FOOTNOTES |
|---|
This work was funded in part by National Heart, Lung, and Blood Institute Grant HL-60666-1 and Children's Hospital and Regional Medical Center Research Fund Grant HR-5836.
Address for reprint requests and other correspondence: X.-H. Ning, Dept. of Pediatrics, Box 356320, Univ. of Washington, 1959 NE Pacific St., Seattle, WA 98195 (E-mail: xh{at}u.washington.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 21, 2001;10.1152/japplphysiol.01035.2001
Received 11 October 2001; accepted in final form 5 December 2001.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Akao, Y,
Otsuki Y,
Kataoka S,
Ito Y,
and
Tsujimoto Y.
Multiple subcellular localization of Bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes.
Cancer Res
54:
2468-2471,
1994.
2.
Armitage, P,
and
Berry G.
Statistical Methods in Medical Research (3rd ed.). Cambridge, UK: Blackwell Science, 1995, p. 358-371.
3.
Bartling, B,
Holtz J,
and
Darmer D.
Contribution of myocyte apoptosis to myocardial infarction.
Basic Res Cardiol
93:
71-84,
1998.
4.
Bialik, S,
Geenen DL,
Sasson IE,
Cheng R,
Horner JW,
Evans SM,
Lord EM,
Koch CJ,
and
Kitsis RN.
Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53.
J Clin Invest
100:
1363-1372,
1997.
5.
Bossenmeyer-Pourie, C,
Koziel V,
and
Daval JL.
Effects of hypothermia on hypoxia-induced apoptosis in cultured neurons from developing rat forebrain: comparison with preconditioning.
Pediatr Res
47:
385-391,
2000.
6.
Breiman, LEO,
Friedman JH,
and
Stone CJ.
Classification and Regression Trees. Pacific Grove, CA: Wadsworth & Brooks, 1985, p. 2-21, 31, 51-54.
7.
Chen, F,
Lu Y,
Zhang Z,
Vallyathan V,
Ding M,
Castranova V,
and
Shi X.
Opposite effect of NF-
B and c-Jun N-terminal kinase on p53-independent Gadd45 induction by arsenite.
J Biol Chem
276:
11414-11419,
2001.
8.
El-Deiry, WS.
Regulation of p53 downstream genes.
Semin Cancer Biol
8:
345-357,
1998.
9.
Finkel, E.
The mitochondrion: is it central to apoptosis?
Science
292:
624-626,
2001.
10.
Fliss, H.
Accelerated apoptosis in reperfused myocardium: friend of foe?
Basic Res Cardiol
93:
90-93,
1998.
11.
Gottlieb, RA,
Burleson KO,
Kloner RA,
Babior BM,
and
Engler RL.
Reperfusion injury induces apoptosis in rabbit cardiomyocytes.
J Clin Invest
94:
1621-1628,
1994.
12.
Jin, S,
Zhao H,
Fan F,
Blanck P,
Fan W,
Colchagie AB,
Fornace AJ,
and
Zhan Q.
BRCA1 activation of the Gadd45 promoter.
Oncogene
19:
4050-4057,
2000.
13.
Kajstura, J,
Cheng W,
Reiss K,
Clark WA,
Sonnenblick EH,
Krajewski S,
Reed JC,
Olivetti G,
and
Anversa P.
Apoptotic and necrotic myocyte cell deaths are independent contributing variable of infarct size in rats.
Lab Invest
74:
86-107,
1996.
14.
Klingenspor, M,
Ivemeyer M,
Wiesinger H,
Haas K,
Heldmaier G,
and
Wiesner RJ.
Biogenesis of thermogenic mitochondria in brown adipose tissue of Djungarian hamsters during cold adaptation.
Biochem J
316:
607-613,
1996.
15.
Koong, AC,
Denko NC,
Hudson KM,
Schindler C,
Swiersz L,
Koch C,
Evans S,
Ibrahim H,
Le QT,
Terris DJ,
and
Giaccia AJ.
Candidate gene for the hypoxic tumor phenotype.
Cancer Res
60:
883-887,
2000.
16.
Krajewski, S,
Tanaka S,
Takayama S,
Schibler MJ,
Fenton W,
and
Reed JC.
Investigation of the subcellular distribution of the Bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes.
Cancer Res
53:
4701-4714,
1993.
17.
Latif, N,
Khan MA,
Briks E,
O'Farrell A,
Westbrook J,
Dunn MJ,
and
Yacoub MH.
Upregulation of the Bcl-2 family of proteins in end stage heart failure.
J Am Coll Cardiol
35:
1769-1777,
2000.
18.
Liu, TJ,
Lai HC,
Wu W,
Chinn S,
and
Wang PH.
Developing a strategy to define the effects of insulin-like growth factor-1 on gene expression profile in cardiomyocytes.
Circ Res
88:
1231-1238,
2001.
19.
Long, X,
Boluyt MO,
Hipolito ML,
Llundberg MS,
Zheng JS,
O'Neill L,
Cirielli C,
Lakatta EG,
and
Crow MT.
p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.
J Clin Invest
99:
2635-2643,
1997.
20.
Lyn, D,
Liu X,
Bennett NA,
and
Emmett NL.
Gene expression profile in mouse myocardium after ischemia.
Physiol Genomics
2:
93-100,
2000.
21.
Maulik, N,
Engelman RM,
Rousou JA,
Flack JE, III,
Deaton D,
and
Das DK.
Ischemic preconditioning reduces apoptosis by upregulating anti-death gene Bcl-2.
Circulation
100:
369-375,
1999.
22.
Mizuno, T,
Miura-Suzuki T,
Yamashita H,
and
Mori N.
Distinct regulation of brain mitochondrial carrier protein-1 and uncoupling protein-2 genes in the rat brain during cold exposure and aging.
Biochem Biophys Res Commun
278:
691-697,
2000.
23.
Ning, XH,
Ding X,
Childs KF,
Bolling SF,
and
Gallagher KP.
Flavone improves functional recovery after ischemia in isolated reperfused rabbit hearts.
J Thorac Cardiovasc Surg
105:
541-549,
1993.
24.
Ning, XH,
Xu CS,
Song YC,
Xiao Y,
and
Portman MA.
Hypothermia preconditioning in isolated rabbit heart (Abstract).
FASEB J
10:
A36,
1996.
25.
Ning, XH,
Xu CS,
Chen SH,
Yao Y,
Song YC,
Li L,
Lupinetti FM,
and
Portman MA.
Application of the "threshold-temperature" (30°C) for ischemic myocardial protection in isolated rabbit hearts (Abstract).
FASEB J
13:
A1064,
1999.
26.
Ning, XH,
Xu CS,
and
Portman MA.
Mitochondrial protein and HSP70 signaling after ischemia in hypothermic-adapted hearts augmented with glucose.
Am J Physiol Regulatory Integrative Comp Physiol
277:
R11-R17,
1999.
27.
Ning, XH,
Xu CS,
Song Y,
Childs KF,
Xiao Y,
Bolling SF,
Lupinetti FM,
and
Portman MA.
Temperature threshold and modulation of energy metabolism in the cardioplegic arrested rabbit heart.
Cryobiology
36:
2-11,
1998.
28.
Ning, XH,
Xu CS,
Song Y,
Xiao Y,
Hu Y,
Lupinetti FM,
and
Portman MA.
Hypothermia preserves function and signaling for mitochondrial biogenesis during subsequent ischemia in isolated rabbit heart.
Am J Physiol Heart Circ Physiol
274:
H786-H793,
1998.
29.
Ning, XH,
Xu CS,
Song Y,
Xiao Y,
Hu Y,
Lupinetti FM,
and
Portman MA.
Temperature threshold and preservation of signaling for mitochondrial membrane proteins during ischemia in rabbit heart.
Cryobiology
36:
321-329,
1998.
30.
Ning, XH,
Zhang J,
Liu J,
Ye Y,
Chen SH,
Form AHL,
Bache RJ,
and
Portman MA.
Signaling and expression for mitochondrial membrane proteins during left ventricular remodeling and contractile failure after myocardial infarction.
J Am Coll Cardiol
32:
282-287,
2000.
31.
Phanithi, PB,
Yoshida Y,
Santana A,
Su M,
Kawamura S,
and
Yasui N.
Mild hypothermia mitigates post-ischemic neuronal death following focal cerebral ischemia in rat brain: immunohistochemical study of Fas, caspase-3 and TUNEL.
Neuropathology
20:
273-282,
2000.
32.
Piot, CA,
Padmanaban D,
Ursell PC,
Sievers RE,
and
Wolfe CL.
Ischemic preconditioning decreases apoptosis in rat hearts in vivo.
Circulation
96:
1598-1604,
1997.
33.
Polyak, K,
Xia Y,
Zweiert JL,
Kinzler KW,
and
Vogelstein B.
A model for p53-induced apoptosis.
Nature
389:
300-305,
1997.
34.
Rees, WD,
Hay SM,
Fontanier-Razzag NC,
Antipatis C,
and
Harries DN.
Expression of the growth arrest genes (GAS and Gadd45) changes during organogenesis in the rat fetus.
J Nutr
129:
1532-1536,
1999.
35.
Russo, T,
Zambrano N,
Esposito F,
Ammendola R,
Cimino F,
Fiscella M,
Jackman J,
O'Connor PM,
Anderson CW,
and
Appella E.
A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress.
J Biol Chem
270:
29386-29391,
1995.
36.
Schmidt-Kastner, R,
Zhao W,
Truettner J,
Belayev L,
Busto R,
and
Ginsberg MD.
Pixel-based image analysis of HSP70, Gadd45 and MAP2 mRNA expression after focal cerebral ischemia: hemodynamic and histological correlates.
Brain Res Mol Brain Res
63:
79-97,
1998.
37.
Velasco-Miguel, S,
Buckbinder L,
Jean P,
Gelbert L,
Talbott R,
Laidlaw J,
Seizinger B,
and
Kley N.
PA26, a novel target of the p53 tumor suppressor and member of the Gadd45 family of DNA damage and growth arrest inducible genes.
Oncogene
18:
127-137,
1999.
This article has been cited by other articles:
|
|
 |
|
  S. Kanemoto, M. Matsubara, M. Noma, B. G. Leshnower, L. M. Parish, B. M. Jackson, R. Hinmon, H. Hamamoto, J. H. Gorman III, and R. C. Gorman Mild Hypothermia to Limit Myocardial Ischemia-Reperfusion Injury: Importance of Timing Ann. Thorac. Surg., January 1, 2009; 87(1): 157 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hamamoto, H. Sakamoto, B. G. Leshnower, L. M. Parish, S. Kanemoto, R. Hinmon, T. Plappert, S. Miyamoto, M. G. St. John-Sutton, J. H. Gorman III, et al. Very Mild Hypothermia During Ischemia and Reperfusion Improves Postinfarction Ventricular Remodeling Ann. Thorac. Surg., January 1, 2009; 87(1): 172 - 177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-H. Ning, E. Y. Chi, N. E. Buroker, S.-H. Chen, C.-S. Xu, Y.-T. Tien, O. M. Hyyti, M. Ge, and M. A. Portman Moderate hypothermia (30{degrees}C) maintains myocardial integrity and modifies response of cell survival proteins after reperfusion Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2119 - H2128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Doyle, K. L. Suchland, T. M.P. Ciesielski, N. S. Lessov, D. K. Grandy, T. S. Scanlan, and M. P. Stenzel-Poore Novel Thyroxine Derivatives, Thyronamine and 3-iodothyronamine, Induce Transient Hypothermia and Marked Neuroprotection Against Stroke Injury Stroke, September 1, 2007; 38(9): 2569 - 2576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-H. Ning, S.-H. Chen, N. E. Buroker, C.-S. Xu, F.-R. Li, S.-P. Li, D.-S. Song, M. Ge, O. M. Hyyti, M. Zhang, et al. Short-cycle hypoxia in the intact heart: hypoxia-inducible factor 1{alpha} signaling and the relationship to injury threshold Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H333 - H341. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. D. Fairchild, I. S. Singh, S. Patel, B. E. Drysdale, R. M. Viscardi, L. Hester, H. M. Lazusky, and J. D. Hasday Hypothermia prolongs activation of NF-{kappa}B and augments generation of inflammatory cytokines Am J Physiol Cell Physiol, August 1, 2004; 287(2): C422 - C431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Kuznetsov, S. Schneeberger, R. Seiler, G. Brandacher, W. Mark, W. Steurer, V. Saks, Y. Usson, R. Margreiter, and E. Gnaiger Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1633 - H1641. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-H. Ning, S.-H. Chen, C.-S. Xu, O. M. Hyyti, K. Qian, J. J. Krueger, and M. A. Portman Hypothermia preserves myocardial function and mitochondrial protein gene expression during hypoxia Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H212 - H219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J Ashton, K. Holmgren, J. Peart, A. R Lankford, G Paul Matherne, S. Grimmond, and J. P Headrick Effects of A1 adenosine receptor overexpression on normoxic and post-ischemic gene expression Cardiovasc Res, March 1, 2003; 57(3): 715 - 726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Valen The basic biology of apoptosis and its implications for cardiac function and viability Ann. Thorac. Surg., February 1, 2003; 75(2): S656 - 660. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
