AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 285: H866-H874, 2003. First published April 24, 2003; doi:10.1152/ajpheart.00596.2002
0363-6135/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/2/H866    most recent
00596.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (22)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hampton, C. R.
Right arrow Articles by Pohlman, T. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hampton, C. R.
Right arrow Articles by Pohlman, T. H.

HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse hearts

Craig R. Hampton,1 Akira Shimamoto,1 Christine L. Rothnie,1 Jeaneatte Griscavage-Ennis,1 Albert Chong,1 David J. Dix,2 Edward D. Verrier,1 and Timothy H. Pohlman1

1Department of Surgery, University of Washington, Seattle, Washington 98104; and 2National Health and Environmental Effects Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Submitted 25 February 2002 ; accepted in final form 22 April 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
We investigated the role of inducible heat shock proteins 70.1 and 70.3 (HSP70.1 and HSP70.3, respectively) in myocardial ischemic preconditioning (IP) in mice. Wild-type (WT) mice and HSP70.1- and HSP70.3-null [HSP70.1/3(–/–)] mice were subjected to IP and examined 24 h later during the late phase of protection. IP significantly increased steady-state levels of HSP70.1 and HSP70.3 mRNA and expression of inducible HSP70 protein in WT myocardium. To assess protection against tissue injury, mice were subjected to 30 min of regional ischemia and 3 h of reperfusion. In WT mice, IP reduced infarct size by 43% compared with sham IP-treated mice. In contrast, IP did not reduce infarct size in HSP70.1/3(–/–) mice. Absence of inducible HSP70.1 and HSP70.3 had no effect, however, on classical or early-phase protection produced by IP, which significantly reduced infarct size in HSP70.1/3(–/–) mice. We conclude that inducible HSP70.1 and HSP70.3 are required for late-phase protection against infarction following IP in mice.

heat shock proteins; knockout mice; reperfusion injury

ISCHEMIC PRECONDITIONING (IP) of the myocardium is a phenomenon whereby brief repetitive periods of transient interruption of coronary blood flow substantially protects the heart against subsequent prolonged ischemia (24). Adaptation of the heart to ischemia following IP is a biphasic phenomenon. There is an early phase of protection that develops within minutes from the initial ischemic insult and lasts 2–3 h, and a late (or delayed) phase that is acquired 24 h later and lasts 3–4 days (reviewed in Ref. 2). In contrast to the early phase of protection, which is effective against myocardial cell death, the second window of protection is effective against both myocardial cell death and myocardial stunning (38). The enhanced resistance to infarction and myocardial stunning afforded by IP and the lasting nature of the response have generated considerable interest in this phenomenon as a potential therapeutic adjunct in the treatment of ischemic heart disease in humans.

It is established that de novo protein synthesis is required for the late phase of IP (34), although the proteins that mediate this cellular resistance to ischemia have not been completely identified. Because increased expression of heat shock proteins (HSPs) in the myocardium increases resistance to ischemia, HSPs are potential mediators of the late phase of IP.

HSPs are highly conserved in eukaryotes (reviewed in Ref. 19), and they are induced by several environmental and intracellular stresses in addition to heat (e.g., cold, exposure to reactive oxygen intermediates, ATP depletion, hyper- or hyposmolarity, and exposure to various toxic substances). Thus HSPs are now generally referred to "stress proteins," and the heat shock response is commonly referred to as the stress response. Stress proteins are named according to their molecular mass (in kDa; e.g., HSP27, HSP60, HSP70, HSP90). In the heart, HSP70 is the primary stress protein responsive to oxidative stress. In mice, the HSP70 family is composed of constitutively expressed (or cognate) proteins designated HSC70 (9) and highly inducible members that are expressed in response to diverse cellular stresses designated HSP70.1 and HSP70.3 (13, 35). HSP synthesis is transcriptionally regulated by a family of transcription factors called heat shock factors (HSFs). Four HSFs have been identified: HSF1, HSF2, HSF3, and HSF4 (25, 26, 33, 36); of these, HSF1 is the most important in the heart. After stress, HSF trimerizes and translocates to the nucleus, where it binds to heat shock element (HSE, a promotor region upstream from the HSP genes) to initiate transcription (23).

The goal of this study was to determine directly whether inducible HSP70.1 and HSP70.3 are required for the late phase of protection following IP of the myocardium. We show that IP increases HSP70.1 and HSP70.3 expression in the heart and that knockout mice, which bear homozygous null alleles for HSP70.1 and HSP70.3, do not acquire IP-induced late-phase tolerance to ischemia. Our results identify a necessary and causal role for HSP70.1 and HSP70.3 in late cardioprotection against infarction following IP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Mice. This study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Washington and with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health [DHEW Publication No. (NIH) 85-23, Revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205]. Before experimentation, all animals were housed in a room with a 71°F temperature, 41% relative humidity, and 12:12-h light-dark cycles in the animal care wing. Animals were allowed access to water and food ad libitum. Wild-type (WT) C57BL/6 mice were purchased from Animal Technologies (Kent, WA).

HSP70.1- and HSP70.3-null mice. Targeted deletion (or gene knockout) of HSP70.1 and HSP70.3 was carried out at the National Health and Environmental Effects Research Laboratory of the US Environmental Protection Agency (EPA; Research Triangle Park, NC) in accordance with the guidelines of the Animal Care and Use Committee of the US EPA and the Guide for the Care and Use of Laboratory Animals. A full description of the generation of these mice is being prepared in a separate article. Briefly, the HSP70.1 and HSP70.3 genes were targeted in AB2.2 embryonic stem cells derived from mouse strain 129Sv/Ev (Lexicon Genetics; Woodlands, TX). The targeting vector was transfected by electroporation, and homologous recombination resulted in a 12-kb deletion of both HSP70.1- and HSP70.3-coding regions as well as insertion of a neomycin-resistance gene. The targeted alleles were confirmed by Southern blot hybridization of genomic DNA from the stem cells. A male chimera derived from microinjection of these targeted stem cells into blastocysts from C57BL/6N mice was bred to C57BL/6N females. Agouti coat color for one male offspring indicated germ-line transmission, and heterozygosity for the HSP70.1- and HSP70.3-targeted allele was confirmed by Southern blot. This F1 resulted in a C57/129 hybrid and was bred back to one of his own heterozygous female offspring to generate homozygous nulls. All data for this article were generated from this line of homozygous null mice. Systolic arterial blood pressure (BP) and heart rate (HR) measurements were performed in conscious restrained mice via tail-cuff plethysmography (Narco Biosystems; Ref. 40). Mice were conditioned twice before measurements were made, and the BP and HR values were recorded as the means ± SE of three separate measurements obtained at each session. HSP70.1- and HSP70.3-null [HSP70.1/3(–/–)] mice had a mean systolic BP of 125 ± 2.2 and a mean HR of 502 ± 10.9 (n = 6), which are comparable to published values of baseline systolic BP and HR for C57BL/6 mice (6, 17).

Surgical preparation. Two anesthetic agents were used for this experiment. For survival experiments, 2,2,2-tribromoethanol (Avertin, Aldrich) was administered before preconditioning [17 ml/g ip of 2.5% solution, initially dissolved in tert-amyl alcohol (Aldrich) and brought to volume in sterile normal saline] to allow rapid and consistent emergence from anesthesia due to its favorable pharmacokinetics. For prolonged ischemia-reperfusion (I/R) experiments, pentobarbital sodium (100 mg/kg ip; Abbott Laboratories) injection was used for anesthesia. In all groups, anesthesia was confirmed by lack of foot-withdrawal reflex.

A midline cervical incision was made and the trachea and larynx were exposed after division of the thyroid isthmus and the strap muscles. Orotracheal intubation was performed with a beveled piece of polyethylene-90 tubing visually confirmed to pass through the exposed trachea until the tip was 2 mm below the larynx. Mechanical ventilation (model 687 mouse ventilator, Harvard Apparatus) was begun with an inspired O2 fraction (FIO2) of 100%, respiratory rate of 85; tidal volume of 0.7 ml, and positive end-expiratory pressure of 3 cm, which was created with a water column connected to the exhaust tube. These ventilator settings were chosen to ensure normal physiological parameters (37) after serial blood gases were obtained in pilot studies. Temperature was monitored via a rectal temperature probe (series 400, YSI) and maintained at 37°C with a heating lamp. A left parasternotomy was performed under the dissecting microscope (Zeiss operating microscope OPMI 6-SDFC) through careful division of three ribs in a cephalocaudal direction parallel to the sternum. The pericardium was reflected thereby exposing the heart. A 7-0 silk suture (US Surgical) on an He-7 needle was passed behind the left anterior descending (LAD) artery just below the left atrial appendage. A Rumel-type snare was created by passing both ends of the suture through the tip of a 22-gauge angiocatheter that could then be tightened and released by sliding a Voss clip down on the angiocatheter for ischemia and reperfusion, respectively. Occlusion-causing ischemia was visually confirmed by blanching of the left ventricle (LV) and subsequent hyperemia with release of the snare. After the experimental protocol was completed, the chest was closed in three layers. The mice were awakened, removed from the ventilator, kept warm with heating blankets, and given 100% FIO2 via nasal cone. Buprenex (Reckitt and Coleman Pharmaceuticals) was given postoperatively for pain control (1 mg/kg ip every 12 h) until the second phase of the experiment.

Experimental protocols. Seventy-eight mice were allocated to six groups, which are schematically represented in Fig. 1. The control group was subjected to 30 min of ischemia followed by3hof reperfusion. Mice in the sham wild-type group underwent thoracotomy identical to the ischemically preconditioned group without occlusion of the LAD, which was followed 24 h later by I/R. Mice in the preconditioned wild-type group (IP) were preconditioned with three cycles of 5 min of ischemia and 5 min of reperfusion followed 24 h later by I/R. HSP70.1/3(–/–) mice were used for the remaining groups. Control HSP70.1/3(–/–) mice were subjected to 30 min of ischemia and 3 h of reperfusion (I/R). Ischemic preconditioning of HSP70.1/3(–/–) mice was carried out with an identical IP regimen of three cycles of 5 min of ischemia and 5 min of reperfusion followed 24 h later by I/R. To analyze the effects of the genetic deletion of HSP70.1 and HSP70.3 on the classic early phase of protection, HSP70.1/3(–/–) mice were subjected to three cycles of alternating 5 min of ischemia and 5 min of reperfusion followed 10 min later by I/R.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1. Schematic of experimental groups, ischemic preconditioning (IP) protocol, and regional ischemia-reperfusion (I/R) injury protocol. IP was carried out with three cycles of 5 min of ischemia followed by 5 min of reperfusion. Delayed "nonclassical" protection from myocardial tissue injury induced by I/R injury was assessed at 24 h after completion of IP. Early or "classical" protection from I/R injury was assessed at 10 min after completion of IP. Sham-treated animals underwent thoracotomy for 30 min without IP. HSP70.1/3(–/–), heat shock protein (HSP)70.1- and HSP70.3-null mice; I, ischemia; R, reperfusion.

 

Of the 78 mice allocated to the experimental groups, 5 were excluded to yield an overall survival of 94%. Four mice (4 of 20) were excluded from the "WT late IP" group due to presumed respiratory failure after extubation. One mouse (1 of 6) died in the "HSP KO acute IP" group from apparent right-heart failure during reperfusion.

Determination of area at risk and infarction. At the completion of the experimental protocol, the aortic root was dissected free from the surrounding fatty tissue to enable cannulation. The LAD artery was then reoccluded, and 4% Evans blue dye was injected into the aortic root to delineate the area at risk (AAR, blue-dye negative), which is the area of myocardium supplied by the LAD artery. The heart was then explanted, rinsed in 0.9% normal saline, and placed in 1% agarose gel (UltraPure agarose, Life Technologies) in PBS (pH 7.4). After the gel solidified, the heart was sectioned parallel to the atrioventricular groove in ~1-mm sections. The sections were incubated at 37°C in 1% triphenyltetrazolium chloride (Sigma) in PBS solution (pH 7.4) for 10 min and then placed in 10% neutral buffered formaldehyde for 24 h. After incubation in triphenyltetrazolium chloride, viable myocardium stains brick red while nonviable (i.e., infarcted) myocardium remains pale (8). After 24 h, the sections were weighed, and each face of every section was photographed with a digital camera (Nikon Coolpix 950) through the Zeiss operating microscope at a constant magnification (approximately x17), and the images were transferred to a computer. The areas (AAR, infarct, and LV) on each face of each section were traced three times and calculated by computer planimetry (Image J version 1.21), and the average value was recorded. These areas were calculated in a blinded fashion by an observer who was unaware of the experimental groups. Infarct volumes were calculated as [(A1 x W1) + (A2 x W2) + (A3 x W3) + (A4 x W4) + (A5 x W5)], where A is the area of infarct for the slice denoted by the subscript, and W is the weight of the respective section. Areas were then expressed as a percentage of the LV or the AAR.

Preparation of nuclear and cytosolic proteins. Nuclear and cytosolic extracts were prepared from murine heart tissue, snap frozen in liquid N2, and maintained at –70°C before use. Tissue was finely ground using a mortar and pestle and was homogenized by 10 strokes of a glass homogenizer in 50% (wt/vol) ice-cold buffer [10 mM HEPES, pH 7.9, that contained 150 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, and 0.6% (vol/vol) Nonidet P-40]. Cytosolic protein was separated from the nuclear pellet in a microcentrifuge at 14,000 rpm for 10 min at 4°C. The nuclear extract was prepared by lysis of the nuclear pellet in high-salt buffer (20 mM HEPES, pH 7.9, that contained 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 2 mM benzamadine, and 5 µg/ml pepstatin A, aprotinin, and leupeptin) followed by centrifugation at 14,000 rpm for 10 min at 4°C. Cytosolic and nuclear fractions were stored at –70°C.

Protein concentrations in the murine heart tissue cytosolic fraction (for Western blot analysis) were determined by the bicinchonic acid method (Pierce; Rockford, IL). Bovine serum albumin was used as the standard.

RT-PCR. RNA transcripts for HSP70.1 and HSP70.3 were assessed 2.5 h after IP or sham IP in wild-type or HSP70.1/3(–/–) mice. Hearts were rapidly explanted and the LV was dissected, rinsed with 0.9% normal saline, snap frozen in liquid N2, and stored at –70°C until subsequent analysis.

RNA was isolated from murine LV using TRIzol reagent (Life Technologies; Gaithersburg, MD) and quantitated by absorption at 260 nm. Expression of mRNA was determined by RT-PCR using primers specific for murine HSP70.1 and HSP70.3 (7). The HSP70.1 amplicon was 285 bp, and the HSP70.3 amplicon was 220 bp. QuantumRNA Classic II18S internal standards (Ambion; Austin, TX) were used to standardize RNA loading differences, which allowed precise quantification of mRNA. Reverse transcription was performed using the SuperScript First-Strand Synthesis System (Life Technologies) with 2 µg of total RNA per cDNA synthesis reaction. The PCR amplification reaction contained 10 mM Tris · HCl (pH 8.4), 1.5 mM MgCl2, 50 mM KCl, 0.4 µMof each primer, 0.2 mM of each deoxynucleotide 5'-triphosphate, 2 µl of cDNA template, and 2.5 U of Taq polymerase (Boehringer Mannheim Biochemicals; Indianapolis, IN). RT-PCR products were electrophoresed on a 2% agarose gel and detected with silver staining. For quantification of band intensity, the optical density of the autoradiograms was determined and normalized to the 18s RNA internal control.

Western immunoblotting analysis. To determine the effects of ischemic (IP) or sham preconditioning on HSP70 myocardial protein content, hearts were explanted immediately following anesthesia (negative control) or 24 h following IP or sham IP. After rapid explantation, the LV was dissected free, rinsed in 0.9% normal saline, snap frozen in liquid N2, and stored at –70°C until subsequent analysis. Cytosolic extracts (20 µg) were separated by SDS-PAGE. The proteins were then transferred (180 mA) to a Hybond-P membrane (Amersham Pharmacia Biotech; Buckinghamshire, UK) for 2 h at 4°C in 25 mM Tris buffer that contained 190 mM glycine and 20% methanol. The membrane was incubated for 1 h at room temperature in bovine lacto transfer technique optimizer [BLOTTO: 20 mM Tris · HCl, pH 7.6, that contained 137 mM NaCl, 0.1% Tween 20, and 5% (wt/vol) Carnation powdered milk], followed by incubation overnight at 4°C with primary antibody that cross-reacts with mouse HSP70 [rabbit anti-human HSP70 (HSP72) polyclonal antibody, 1:20,000 dilution in buffer; StressGen; Victoria, BC, Canada]. The non-binding primary antibody was washed out, and the membrane was incubated with secondary antibody (anti-rabbit IgG-horseradish peroxidase; Santa Cruz Biotechnology; Santa Cruz, CA) for 1 h at 4°C. HSP70 was detected by autoradiography using SuperSignal West Pico Substrate (Pierce) for chemiluminescent detection of horseradish peroxidase.

Statistical analysis. Data are reported as means ± SE. Comparisons between groups were analyzed using a one-way ANOVA. If the ANOVA showed an overall difference, post hoc contrasts were performed with Student's t-tests for unpaired data. Statistical differences were considered significant for P values <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Expression of HSP70.1 and HSP70.3 mRNA. To determine the effects of ischemic preconditioning on changes in steady-state levels for HSP70 mRNA, the accumulations of myocardial HSP70.1- and HSP70.3-specific transcripts were assessed by relative RT-PCR 2.5 h after mice were treated by IP. As shown in Fig. 2A, sham preconditioning, which exposed the animals to the stress of the thoracotomy but not the stress of coronary artery occlusion, did not induce accumulation of HSP70.1 mRNA. However, IP of wild-type mice resulted in the appearance of HSP70.1 mRNA at 2.5 h after completion of the IP regimen. IP induced a 2.3-fold rise in steady-state levels of HSP70.1 mRNA (P < 0.0005 compared with other groups). Predictably, IP of HSP70.1/3(–/–) mice did not induce detectable HSP70.1 mRNA levels, which is consistent with the presence of a null allele at this genetic locus. A similar and more robust response was observed in HSP70.3 mRNA induction by IP (Fig. 2B). Compared with sham preconditioning, which did not lead to accumulation of transcripts of HSP70.3, IP of wild-type mice significantly increased steady-state levels of HSP70.3 mRNA and resulted in a 3.6-fold increase above undetectable levels in sham preconditioned mice or unconditioned wild-type mice (P < 0.003 compared with other groups). IP of HSP70.1/3(–/–) mice failed to induce HSP70.3 mRNA accumulation, which is also consistent with the presence of a null allele at this genetic locus.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Relative RT-PCR for HSP70.1 and HSP70.3 mRNAs detected by silver staining and quantified by densitometry (see MATERIALS AND METHODS). A: RT-PCR for HSP70.1 mRNA was carried out on myocardial samples that were obtained from homogenized left ventricles (LVs) 90 min after wild-type (WT) mice were treated by either IP (n = 4) or by sham IP (n = 3), or 90 min after HSP70.1/3(–/–) (HSP70 KO) mice were treated by IP (n = 4). The 18s rRNA is shown as an internal control to normalize RNA loading differences between lanes. B: densitometry for the gel depicted in A was done on each lane in each group, normalized to 18s, and averaged for comparison between groups (*P < 0.0005, WT + IP vs. WT + sham IP). Image in A and densitometric analysis in B are representative of three separate experiments. C: RT-PCR for HSP70.3 mRNA was carried out on similar myocardial samples described for A. D: densitometric analysis, which was performed as described for B. **P < 0.003, WT + IP vs. WT + sham IP, and is representative of three separate experiments.

 

Expression of HSP70 protein in cytoplasm. To determine the effects of IP or sham IP on myocardial HSP70 protein content, HSP70 protein expression was examined at 24 h after preconditioning by Western blotting of cytosolic proteins (Fig. 3, A and B) as outlined in MATERIALS AND METHODS. Consistent with increases in steady-state levels of HSP70 mRNA, IP of wild-type mice significantly increased HSP70 protein expression compared with sham preconditioned wild-type mice that underwent thoracotomy alone (2.7 ± 0.4-fold increase in IP-treated mice vs. 1.3 ± 0.4-fold increase in sham preconditioned wild-type mice; P < 0.03). As expected, IP treatment of HSP70.1/3(–/–) mice did not produce a change in myocardial HSP70 protein content.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 3. Western blot for myocardial HSP70 protein expression induced by IP. A: myocardial samples were obtained from the LV 24 h after IP treatment (described in MATERIALS AND METHODS) of either a WT (WT IP) or HSP70.1/3(–/–) mouse [HSP70.1/3(–/–)IP] and from the LV of a WT mouse either immediately after thoracotomy (WT control) or after thoracotomy and 30 min of sham preconditioning (sham IP). Protein samples were probed for inducible HSP70 with a primary antibody that cross-reacts with mouse HSP70 [rabbit anti-human HSP70 (HSP72) polyclonal antibody]. Recombinant human HSP70 (HSP72; inducible HSP70) was loaded into the first lane of the gel. Similar blots were obtained in additional separate experiments [WT IP, n = 6; HSP70.1/3(–/–)IP, n = 6; WT control, n = 3; and sham IP, n = 6]. B: Western blot is semiquantified through densitometric analysis of the autoradiograms and averaged with expression of HSP70 set relative to the negative control, which is arbitrarily set at 1 unit. Values are means ± SE; *P < 0.03 vs. all other groups.

 

AAR and infarct-size determination. In all groups, the AAR comprised 45–53% of the LV (Fig. 4A) and was not statistically different between groups. Treatment of wild-type mice with IP 24 h before I/R significantly reduced infarction size by 43% (expressed as a percentage of AAR) compared with sham preconditioned wild-type mice (17.7 ± 1.8 for wild-type IP vs. 31.1 ± 5.0% for wild-type sham mice; P < 0.03; Fig. 4B, solid bars). Notably, thoracotomy alone in wild-type mice without IP (sham IP-treated mice) did not effect subsequent infarct size after 30 min of ischemia and 3 h of reperfusion compared with mice subjected only to the 30-min, 3-h I/R injury alone [31.1 ± 5.0 vs. 30.6 ± 3.1%; P = not significant (NS)]. Thus the effect of IP on reduction in infarct size in these experiments was due only to the effects of brief periods of I/R during the preconditioning period and was not due to the surgical stress of thoracotomy.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 4. Effects of IP on infarct size in WT and HSP70.1/3(–/–) mice subjected to I/R. A: area at risk (AAR), expressed as a percentage of the LV, was comparable in all groups and comprised 45–53% of the LV. B: infarction is expressed as a percentage of the AAR for WT mice subjected to I/R, I/R 24 h after IP, or I/R 24 h after sham IP and for HSP70.1/3(–/–) mice subjected to I/R, I/R 24 h after IP, or I/R 10 min after IP. *P < 0.03 vs. all other groups.

 

Deletion of HSP70.1 and HSP70.3 in knockout mice had no effect on infarct size compared with wild-type mice after 30 min of regional ischemia and 3 h of reperfusion (27.4 ± 2.1 vs. 30.6 ± 3.1%; P = NS). In contrast with wild-type mice, however, IP of mice bearing homozygous null alleles for inducible HSP70.1 and HSP70.3 failed to induce late-phase cardioprotection. Infarct size in HSP70.1/3(–/–) mice that had undergone IP 24 h before I/R was the same as infarct size in HSP70.1/3(–/–) mice subjected to I/R without prior IP (31.0 ± 2.2 vs. 27.4 ± 2.1%; P = NS; Fig. 4B, gray bars).

To determine whether inducible HSP70.1 and HSP70.3 mediated the classic or early phase of cardioprotection produced by IP, HSP70.1/3(–/–) mice were subjected to 30 min of ischemia and 3 h of reperfusion 10 min after completing IP. As shown in Fig. 4B (gray bars), the early phase of protection following IP was preserved in HSP70.1/3(–/–) mice. IP significantly reduced infarct size (38%) compared with control HSP70.1/3(–/–) mice (16.9 ± 1.9 vs. 27.4 ± 2.1%; P < 0.003) during the early phase, which indicates that targeted deletion of HSP70.1 and HSP70.3 affected only the delayed phase of IP-induced protection.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The findings of this study are that IP induces an increase in levels of HSP70.1 and HSP70.3 steady-state mRNA, which is followed by the increased expression of HSP70.1 and HSP70.3 proteins, and that IP-induced expression of HSP70.1 and HSP70.3 is associated with the development of delayed tolerance to tissue injury produced by myocardial I/R. Furthermore, we demonstrate that targeted deletion of the HSP70.1 and HSP70.3 genes in mice abolishes the late infarct-sparing effect of IP; in contrast, the protective effects of early (classic) IP are preserved in the HSP70.1/3(–/–) mice. Taken together, our data indicate that inducible HSP70.1 and HSP70.3 are required for acquisition of late but not early protection against myocardial infarction following IP in a mouse model of injury.

Previous observations have suggested that expression of inducible forms of HSP70 protects the myocardium against oxidative stress including I/R injury. For example, transgenic overexpression of HSP70 in the myocardium confers protection against both myocardial stunning and infarction following ischemia (14, 21, 28, 30, 42), whereas pharmacological induction of HSP70 in the myocardium also imparts resistance to ischemia (15, 20, 29, 46). Furthermore, heat stress induction of HSP70 in the myocardium is associated with the development of "cross tolerance" to subsequent prolonged ischemia (4, 43). Thus numerous studies substantiate the conclusion that cellular expression of HSP70 can confer protection against I/R injury in the myocardium. Because increased expression of inducible HSP70 mimics the cardioprotective effects of IP, these studies also suggest that inducible HSP70 may mediate the late protein synthesis-dependent phase of IP in the heart.

Although IP of the myocardium has been shown to increase the expression of inducible HSP70 in the myocardium and is associated with cardioprotection (4, 16, 22, 27, 44), no direct evidence has been reported to support the conclusion that HSP70 mediates the late cardioprotection induced by IP. Moreover, two previous studies suggest that expression of inducible HSP70 following IP does not provide protection against ischemia, as assessed by a reduction in infarct size. Using a rat model of myocardial I/R, Qian et al. (32) reported that IP, despite enhancing expression of myocardial HSP72, did not provide late protection against infarction following regional ischemia. Also, Tanaka et al. (39) observed that IP failed to reduce infarction after prolonged ischemia in rabbits despite increased expression of HSP72–73 induced by IP. These discordant results might be explained at least in part by differences in species, preconditioning regimens, or quantitative differences in the cellular expression of inducible HSP70. Furthermore, in both of these experiments, the IP regimens that were used failed to produce delayed protection against infarction. Therefore, whether inducible HSP70 is required for delayed IP-induced protection cannot be assessed in these two studies.

The mechanism by which HSP70 provides cellular protection against oxidative, infectious, thermal, and other stresses in the myocardium has been reviewed in detail (1). HSP70 in the myocardium primarily functions as a molecular chaperone by facilitating the folding, assembly, and disassembly of other proteins. Because protein folding is a requisite of final protein structure and thus function, chaperones play a critical role in cellular homeostasis under physiological conditions and during cellular stress. The specific cellular functions of chaperones include solubilization of denatured protein aggregates, facilitation of the restoration of the function of renatured proteins, active disassembly of clathrin-coated vesicles, facilitation of intracellular trafficking of polypeptide chains across organelle membranes, and transportation of irreversibly damaged proteins to degradative organelles and proteasomes.

Little is known about the mechanisms by which the HSPs protect the myocardium against oxidative stress, although, given the diverse functions of HSPs, protection of cardiomyocytes may result from several mechanisms as outlined above. Alternatively, a recent study with confocal microscopy demonstrated that heat shock-induced stress-protein expression in rat heart increases HSP70 expression in microvascular blood vessels and not in cardiomyocytes (18). This study suggests that the cross tolerance to I/R injury after heat shock, mediated by HSP70 expression in the myocardium, may involve alterations in microvascular blood flow during ischemia or following restoration of flow to ischemic areas during reperfusion. However, it is still possible that IP-induced HSP70 expression is localized to cardiomyocytes, and that the tolerance to I/R injury mediated by HSP70 expression in the myocardium after IP is primarily due to intracellular mechanisms in cardiomyocytes mediated by HSP70.

In addition to its general chaperone functions, HSP70 may provide cellular protection against I/R injury through other mechanisms that are specific to oxidative stress. Thus in heat-stressed animal hearts, preservation of myocardial mitochondrial structure (3) and function (31, 45) and conservation of levels of ATP and creatine phosphate have been observed following I/R injury (45). These improvements in energy metabolism with heat shock treatment of hearts before I/R are associated with enhanced recovery in myocardial performance and reduced I/R injury. Also, a decrease in the duration of cardiomyocyte action potentials secondary to an increase in activity of ATP-sensitive potassium channels (12, 27) and diminished accumulation of intracellular Ca2+ (45) with resulting reduced Ca2+ sensitivity (22) have been reported to be induced by heat stress and to limit I/R injury.

We have used a mouse model of in vivo open-chest ischemic preconditioning and I/R to study the molecular mediators of protection. Although it was suggested that open-chest models requiring thoracotomy may induce sufficient stress to confound results (41), our data do not support this with respect to infarction. Sham preconditioned animals subjected only to general anesthesia and a paramedian sternotomy were not protected against infarction. Moreover, sham IP had no effect on HSP70.1 and HSP70.3 mRNA or protein expression but did increase HSF activation in the nucleus compared with negative controls (unpublished observation), an event that has been shown to be insufficient to result in HSP70 translation (5). These data suggest that in an open-chest mouse model, sham operation may induce a minor stress that initiates cell signaling pathways but is insufficient to induce de novo HSP70.1 or HSP70.3 transcription or translation.

In conclusion, we have directly demonstrated a causal role for inducible HSP70.1 and HSP70.3 in the late phase of protection against infarction following IP in a mouse model of in vivo injury. These data provide additional evidence that the mechanism of late protection following IP is a complex phenomenon that involves numerous critical proteins (10, 11) including inducible HSP70.1 and HSP70.3.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported in part by the National Heart, Lung, and Blood Institute (NHLBI) Grant R01 HL-61767 and NHLBI Cardiovascular Training Grant 5T32 HL-07828 (to C. R. Hampton). Furthermore, the information in this document has been funded in part by the US Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Nigel Mackman of Scripps Research Institute for a critical review of this paper.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. R. Hampton, 1959 N.E. Pacific St., Box 356410, Univ. of Washington, Seattle, WA 98195 (E-mail: champton{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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Benjamin IJ and McMillan DR. Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res 83: 117–132, 1998.[Abstract/Free Full Text]
  2. Bolli R. The late phase of preconditioning. Circ Res 87: 972–983, 2000.[Abstract/Free Full Text]
  3. Currie RW, Karmazyn M, Kloc M, and Mailer K. Heat-shock response is associated with enhanced postischemic ventricular recovery. Circ Res 63: 543–549, 1988.[Abstract/Free Full Text]
  4. Currie RW, Tanguay RM, and Kingma JG Jr. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 87: 963–971, 1993.[Abstract/Free Full Text]
  5. Davidson S, Hoj P, Gabriele T, and Anderson RL. In vivo growth of a murine lymphoma cell line alters regulation of expression of HSP72. Mol Cell Biol 15: 1071–1078, 1995.[Abstract]
  6. Desai KH, Sato R, Schauble E, Barsh GS, Kobilka BK, and Bernstein D. Cardiovascular indexes in the mouse at rest and with exercise: new tools to study models of cardiac disease. Am J Physiol Heart Circ Physiol 272: H1053–H1061, 1997.[Abstract/Free Full Text]
  7. Dix DJ, Garges JB, and Hong RL. Inhibition of HSP70–1 and HSP70–3 expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic. Mol Reprod Dev 51: 373–380, 1998.[Web of Science][Medline]
  8. Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K, Mercier JC, Corday E, and Ganz W. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J 101: 593–600, 1981.[Web of Science][Medline]
  9. Giebel LB, Dworniczak BP, and Bautz EK. Developmental regulation of a constitutively expressed mouse mRNA encoding a 72-kDa heat shock-like protein. Dev Biol 125: 200–207, 1988.[Web of Science][Medline]
  10. Guo Y, Bao W, Wu WJ, Shinmura K, Tang XL, and Bolli R. Evidence for an essential role of cyclooxygenase-2 as a mediator of the late phase of ischemic preconditioning in mice. Basic Res Cardiol 95: 479–484, 2000.[Web of Science][Medline]
  11. Guo Y, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, and Bolli R. The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci USA 96: 11507–11512, 1999.[Abstract/Free Full Text]
  12. Hoag JB, Qian YZ, Nayeem MA, D'Angelo M, and Kukreja RC. ATP-sensitive potassium channel mediates delayed ischemic protection by heat stress in rabbit heart. Am J Physiol Heart Circ Physiol 273: H2458–H2464, 1997.[Abstract/Free Full Text]
  13. Hunt CR, Gasser DL, Chaplin DD, Pierce JC, and Kozak CA. Chromosomal localization of five murine HSP70 gene family members: Hsp70–1, Hsp70–2, Hsp70–3, Hsc70t, and Grp78. Genomics 16: 193–198, 1993.[Web of Science][Medline]
  14. Jayakumar J, Suzuki K, Sammut IA, Smolenski RT, Khan M, Latif N, Abunasra H, Murtuza B, Amrani M, and Yacoub MH. Heat shock protein 70 gene transfection protects mitochondrial and ventricular function against ischemia-reperfusion injury. Circulation 104, Suppl 1: I303–I307, 2001.
  15. Katori M, Tamaki T, Takahashi T, Tanaka M, Kawamura A, and Kakita A. Prior induction of heat shock proteins by a nitric oxide donor attenuates cardiac ischemia/reperfusion injury in the rat. Transplantation 69: 2530–2537, 2000.[Web of Science][Medline]
  16. Knowlton AA, Brecher P, and Apstein CS. Rapid expression of heat shock protein in the rabbit after brief cardiac ischemia. J Clin Invest 87: 139–147, 1991.[Web of Science][Medline]
  17. Krege JH, Hodgin JB, Hagaman JR, and Smithies O. A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 25: 1111–1115, 1995.[Abstract/Free Full Text]
  18. Leger JP, Smith FM, and Currie RW. Confocal microscopic localization of constitutive and heat shock-induced proteins HSP70 and HSP27 in the rat heart. Circulation 102: 1703–1709, 2000.[Abstract/Free Full Text]
  19. Lindquist S. The heat-shock response. Annu Rev Biochem 55: 1151–1191, 1986.[Web of Science][Medline]
  20. Lubbers NL, Polakowski JS, Wegner CD, Burke SE, Diaz GJ, Daniell KM, and Cox BF. Oral bimoclomol elevates heat shock protein 70 and reduces myocardial infarct size in rats. Eur J Pharmacol 435: 79–83, 2002.[Web of Science][Medline]
  21. Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, and Dillmann WH. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 95: 1446–1456, 1995.[Web of Science][Medline]
  22. Marber MS, Walker JM, Latchman DS, and Yellon DM. Attenuation by heat stress of a submaximal calcium paradox in the rabbit heart. J Mol Cell Cardiol 25: 1119–1126, 1993.[Web of Science][Medline]
  23. Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 12: 3788–3796, 1998.[Free Full Text]
  24. Murry CE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124–1136, 1986.[Abstract/Free Full Text]
  25. Nakai A and Morimoto RI. Characterization of a novel chicken heat shock transcription factor, heat shock factor 3, suggests a new regulatory pathway. Mol Cell Biol 13: 1983–1997, 1993.[Abstract/Free Full Text]
  26. Nakai A, Tanabe M, Kawazoe Y, Inazawa J, Morimoto RI, and Nagata K. HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Mol Cell Biol 17: 469–481, 1997.[Abstract]
  27. Okubo S, Bernardo NL, Elliott GT, Hess ML, and Kukreja RC. Tyrosine kinase signaling in action potential shortening and expression of HSP72 in late preconditioning. Am J Physiol Heart Circ Physiol 279: H2269–H2276, 2000.[Abstract/Free Full Text]
  28. Okubo S, Wildner O, Shah MR, Chelliah JC, Hess ML, and Kukreja RC. Gene transfer of heat-shock protein 70 reduces infarct size in vivo after ischemia/reperfusion in the rabbit heart. Circulation 103: 877–881, 2001.[Abstract/Free Full Text]
  29. Ooie T, Takahashi N, Saikawa T, Nawata T, Arikawa M, Yamanaka K, Hara M, Shimada T, and Sakata T. Single oral dose of geranylgeranylacetone induces heat-shock protein 72 and renders protection against ischemia/reperfusion injury in rat heart. Circulation 104: 1837–1843, 2001.[Abstract/Free Full Text]
  30. Plumier JC, Ross BM, Currie RW, Angelidis CE, Kazlaris H, Kollias G, and Pagoulatos GN. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest 95: 1854–1860, 1995.[Web of Science][Medline]
  31. Polla BS, Kantengwa S, Francois D, Salvioli S, Franceschi C, Marsac C, and Cossarizza A. Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. Proc Natl Acad Sci USA 93: 6458–6463, 1996.[Abstract/Free Full Text]
  32. Qian YZ, Bernardo NL, Nayeem MA, Chelliah J, and Kukreja RC. Induction of 72-kDa heat shock protein does not produce second window of ischemic preconditioning in rat heart. Am J Physiol Heart Circ Physiol 276: H224–H234, 1999.[Abstract/Free Full Text]
  33. Rabindran SK, Giorgi G, Clos J, and Wu C. Molecular cloning and expression of a human heat shock factor, HSF1. Proc Natl Acad Sci USA 88: 6906–6910, 1991.[Abstract/Free Full Text]
  34. Rizvi A, Tang XL, Qiu Y, Xuan YT, Takano H, Jadoon AK, and Bolli R. Increased protein synthesis is necessary for the development of late preconditioning against myocardial stunning. Am J Physiol Heart Circ Physiol 277: H874–H884, 1999.[Abstract/Free Full Text]
  35. Sarge KD, Zimarino V, Holm K, Wu C, and Morimoto RI. Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. Genes Dev 5: 1902–1911, 1991.[Abstract/Free Full Text]
  36. Schuetz TJ, Gallo GJ, Sheldon L, Tempst P, and Kingston RE. Isolation of a cDNA for HSF2: evidence for two heat shock factor genes in humans. Proc Natl Acad Sci USA 88: 6911–6915, 1991.[Abstract/Free Full Text]
  37. Siemann DW and Hill RP. Quantitative changes in the arterial blood gases of mice following localized irradiation of the lungs. Radiat Res 93: 560–566, 1983.[Web of Science][Medline]
  38. Sun JZ, Tang XL, Knowlton AA, Park SW, Qiu Y, and Bolli R. Late preconditioning against myocardial stunning. An endogenous protective mechanism that confers resistance to postischemic dysfunction 24 h after brief ischemia in conscious pigs. J Clin Invest 95: 388–403, 1995.[Web of Science][Medline]
  39. Tanaka M, Fujiwara H, Yamasaki K, Miyamae M, Yokota R, Hasegawa K, Fujiwara T, and Sasayama S. Ischemic preconditioning elevates cardiac stress protein but does not limit infarct size 24 or 48 h later in rabbits. Am J Physiol Heart Circ Physiol 267: H1476–H1482, 1994.[Abstract/Free Full Text]
  40. Thomas SE, Anderson S, Gordon KL, Oyama TT, Shankland SJ, and Johnson RJ. Tubulointerstitial disease in aging: evidence for underlying peritubular capillary damage, a potential role for renal ischemia. J Am Soc Nephrol 9: 231–242, 1998.[Abstract]
  41. Triana JF, Li XY, Jamaluddin U, Thornby JI, and Bolli R. Postischemic myocardial "stunning." Identification of major differences between the open-chest and the conscious dog and evaluation of the oxygen radical hypothesis in the conscious dog. Circ Res 69: 731–747, 1991.[Abstract/Free Full Text]
  42. Trost SU, Omens JH, Karlon WJ, Meyer M, Mestril R, Covell JW, and Dillmann WH. Protection against myocardial dysfunction after a brief ischemic period in transgenic mice expressing inducible heat shock protein 70. J Clin Invest 101: 855–862, 1998.[Web of Science][Medline]
  43. Yamashita N, Hoshida S, Taniguchi N, Kuzuya T, and Hori M. Whole-body hyperthermia provides biphasic cardioprotection against ischemia/reperfusion injury in the rat. Circulation 98: 1414–1421, 1998.[Abstract/Free Full Text]
  44. Yang XM, Baxter GF, Heads RJ, Yellon DM, Downey JM, and Cohen MV. Infarct limitation of the second window of protection in a conscious rabbit model. Cardiovasc Res 31: 777–783, 1996.[Web of Science][Medline]
  45. Yellon DM, Pasini E, Cargnoni A, Marber MS, Latchman DS, and Ferrari R. The protective role of heat stress in the ischaemic and reperfused rabbit myocardium. J Mol Cell Cardiol 24: 895–907, 1992.[Web of Science][Medline]
  46. Zhou JJ, Pei JM, Wang GY, Wu S, Wang WP, Cho CH, and Wong TM. Inducible HSP70 mediates delayed cardioprotection via U-50488H pretreatment in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 281: H40–H47, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Hu, X. Zhu, J. R. Triggs, Y. Tao, Y. Wang, L. Lichtenstein, M. Bissonnette, M. W. Musch, and E. B. Chang
Inflammation-induced, 3'UTR-dependent translational inhibition of Hsp70 mRNA impairs intestinal homeostasis
Am J Physiol Gastrointest Liver Physiol, May 1, 2009; 296(5): G1003 - G1011.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
Y. Tao, J. Hart, L. Lichtenstein, L. J. Joseph, M. J. Ciancio, S. Hu, E. B. Chang, and M. Bissonnette
Inducible heat shock protein 70 prevents multifocal flat dysplastic lesions and invasive tumors in an inflammatory model of colon cancer
Carcinogenesis, January 1, 2009; 30(1): 175 - 182.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
A. Redel, V. Jazbutyte, T. M. Smul, M. Lange, T. Eckle, H. Eltzschig, N. Roewer, and F. Kehl
Impact of Ischemia and Reperfusion Times on Myocardial Infarct Size in Mice In Vivo
Experimental Biology and Medicine, January 1, 2008; 233(1): 84 - 93.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
Y.-K. Kim, J. Suarez, Y. Hu, P. M. McDonough, C. Boer, D. J. Dix, and W. H. Dillmann
Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy
Circulation, June 6, 2006; 113(22): 2589 - 2597.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
I. E. Konstantinov, S. Arab, J. Li, J. G. Coles, C. Boscarino, A. Mori, E. Cukerman, F. Dawood, M. M.H. Cheung, M. Shimizu, et al.
The remote ischemic preconditioning stimulus modifies gene expression in mouse myocardium
J. Thorac. Cardiovasc. Surg., November 1, 2005; 130(5): 1326 - 1332.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
S. J. Schomisch, D. G. Murdock, N. Hedayati, J. L. Carino, E. J. Lesnefsky, and B. L. Cmolik
Cardioplegia prevents ischemia-induced transcriptional alterations of cytoprotective genes in rat hearts: A DNA microarray study
J. Thorac. Cardiovasc. Surg., October 1, 2005; 130(4): 1151 - 1151.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. Zhang, Z. Xu, X.-R. He, L. H. Michael, and C. Patterson
CHIP, a cochaperone/ubiquitin ligase that regulates protein quality control, is required for maximal cardioprotection after myocardial infarction in mice
Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2836 - H2842.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. Kubulus, F. Roesken, M. Amon, M. Rucker, M. Bauer, I. Bauer, and M. D. Menger
Mechanism of the delay phenomenon: tissue protection is mediated by heme oxygenase-1
Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2332 - H2340.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
M. Yada, A. Shimamoto, C. R. Hampton, A. J. Chong, H. Takayama, C. L. Rothnie, D. J. Spring, H. Shimpo, I. Yada, T. H. Pohlman, et al.
FR167653 diminishes infarct size in a murine model of myocardial ischemia-reperfusion injury
J. Thorac. Cardiovasc. Surg., October 1, 2004; 128(4): 588 - 594.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
A. J. Chong, A. Shimamoto, C. R. Hampton, H. Takayama, D. J. Spring, C. L. Rothnie, M. Yada, T. H. Pohlman, and E. D. Verrier
Toll-like receptor 4 mediates ischemia/reperfusion injury of the heart
J. Thorac. Cardiovasc. Surg., August 1, 2004; 128(2): 170 - 179.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/2/H866    most recent
00596.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (22)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hampton, C. R.
Right arrow Articles by Pohlman, T. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hampton, C. R.
Right arrow Articles by Pohlman, T. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.