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Cardiovascular Research 2003 59(2):450-459; doi:10.1016/S0008-6363(03)00399-7
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Copyright © 2003, European Society of Cardiology

Brief episode of ischemia activates protective genetic program in rat heart: a gene chip study

Boris Z. Simkhovicha,b,*, Paul Marjoramc, Coralie Poizatd,e, Larry Kedesb,d,e and Robert A. Klonera,b

aHeart Institute, Good Samaritan Hospital, Los Angeles, CA 90017, USA
bDepartment of Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA
cDepartment of Preventive Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA
dDepartment of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA
eInstitute for Genetic Medicine, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA

* Corresponding author. Heart Institute, Good Samaritan Hospital, 1225 Wilshire Boulevard, Los Angeles, CA 90017, USA. Tel.: +1-213-977-4194; fax: +1-213-977-4107. simkhovi{at}hsc.usc.edu

Received 24 January 2003; revised 10 April 2003; accepted 29 April 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Brief episodes of ischemia of 20 min or less have the potential to protect the heart. Such episodes are associated primarily with reversible ischemic injury yet they induce changes in gene expression. The purpose of the study was to determine whether activation of protective genes takes place within 4 h following a brief episode of ischemia that would mimic angina pectoris. Methods: Three groups of rats were studied. In the control (Ctrl) group, hearts were immediately excised following anesthesia; in the sham-operated (SO) group, opened-chest rats received 4 h and 20 min of no intervention; and in the group subjected to ischemia (SI) hearts received 20 min of proximal coronary occlusion followed by 4 h of reperfusion. Hearts from the SI group were divided into nonischemic (NI) and ischemic (Isc) areas. Changes in gene expression pattern were analyzed by using Affymetrix Gene Chips. Results: Ischemia led to strong upregulation of mRNA transcripts for heat shock proteins 70, 27, 105, 86 and 40 kDa, vascular endothelial growth factor, brain-derived neurotrophic factor, plasminogen activator inhibitor-1, activating transcription factor 3, B-cell translocation gene 2, and growth arrest and DNA damage inducible 45 {alpha} protein compared to the NI tissue. The majority of mRNAs whose levels increased following brief ischemia were of a protective nature. Conclusion: Genetic reprogramming emerging during or following brief episodes of ischemia that simulate angina, can be characterized as protective in nature. Developing new therapeutic strategies aimed to promote this protective response represents a legitimate target for future research.

KEYWORDS Heart; Gene expression; Ischemia; Preconditioning


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Brief episodes of ischemia of 20 min or less are associated primarily with reversible ischemic injury rather than necrosis and mimic to some extent the ischemic insult associated with angina pectoris. Such episodes of ischemia may actually precondition the heart against future ischemic insult. Numerous cellular factors representing regulatory pathways (i.e. transcription factors, proinflammatory proteins, early response genes, growth and apoptosis related factors and heat shock proteins) have been reported to be involved and/or affected by discrete episodes of ischemia [1–6]. Such changes could be occurring at one or more molecular steps including alterations in rates of gene transcription or pre-mRNA processing, mRNA stability, mRNA translation or protein turnover. The development of high-density gene microarrays (generally referred to as ‘gene chips’) have provided researchers a unique opportunity to study the relative levels of gene transcripts for thousands of genes simultaneously in the same tissue sample. So far, several studies that have employed this innovative technology in animal models have focused their attention primarily on gene expression changes during the postmyocardial infarction period including changes associated with the remodeling process [7–10]. However, changes that occur following the early stages of myocardial ischemia, such as might occur following a simulated episode of angina also may be important. Heart adaptation to ischemic stress (both immediate and delayed) is induced during brief episodes of ischemia [11,12]. We sought to determine whether the brief episode of ischemia induces changes in levels of mRNAs encoding proteins that might be protective and if so, which specific transcripts were increased when a broad array were examined. For these purposes, we employed an in vivo model of rat heart ischemia of 20-min duration followed by 4 h of reperfusion to allow enough time for any changes in transcription or mRNA stability to affect mRNA levels. We assayed the mRNAs of 8800 genes using an Affymetrix Gene Chips (Affymetrix Santa Clara, CA, USA).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The study conforms to the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised, 1996).

2.1 Surgical preparation and experimental protocols
Spraque–Dawley female rats, 8–9 months old (n = 18) were divided into three groups; control (Ctrl), sham-operated (SO) and subjected to ischemia (SI) groups. There were four rats in the Ctrl, four rats in the SO and ten rats in the SI groups. All animals were anesthetized with a mixture of ketamine and xylazine given i.p. in doses of 60 and 10 mg/kg, respectively. In the Ctrl group hearts were immediately excised following anesthesia and frozen in liquid nitrogen. In the SO and SI groups the anesthesia was maintained with an additional dose of anesthetics (1/4 of the initial), administered every hour. In both groups, a suture was passed around the left coronary artery, and animals from the SO group received 4 h and 20 min of no intervention period. Hearts in the SO group were excised and frozen in liquid nitrogen. The SI group received 20 min of coronary occlusion, followed by 4 h of reperfusion. At the end of the protocol, the left coronary artery in the SI group was briefly reoccluded, and Unisperse Blue Dye (Ciba-Geigy, Hawthorne, NJ, USA) was injected via a catheter inserted into the left carotid artery. Since it has been shown that the process of sacrificing the animals may significantly alter the expression pattern [13], special care was taken to minimize variability during this stage of experiment. For this purpose all animals were given 2 mEquiv. of KCl i.v., which instantly caused cessation of cardiac activity, after which the hearts were removed and placed in liquid nitrogen. Half of the animals were used for the Gene Chip analysis, the rest for the Northern blots analysis. In each group frozen left ventricles were separated from the rest of the heart and great arteries and pooled together. Based on the distribution of the Blue Dye, the left ventricles in the SI group were further separated into the ischemic (Isc) and the nonischemic (NI) tissues. Thus from three experimental groups we obtained four pooled samples; representing the Ctrl and SO hearts and the Isc and the NI tissues as well. For the purposes of our study two sets of pooled samples were obtained. One set of four samples was used in Gene Chip, and the other in the Northern Blot analysis.

2.2 RNA isolation, probe preparation, and Affymetrix gene chip array analysis
Total RNA was extracted from pooled Ctrl, SO, Isc and NI samples by using TRIzol reagent (Life Technologies, Rockville, MD, USA). Probe preparation and hybridization with Rat Gene Array U34A (Affymetrix) were carried out according to protocols recommended by Affymetrix. Hybridizations were conducted on a one array per one pooled sample basis and were replicated. Thus we had four tissue aliquots and four arrays in the first hybridization, and four new tissue samples and a new set of four arrays in the second one. In other words, Gene Chip experiments were carried out as biological replicates; i.e. the same animals were sampled twice. Altogether we used eight arrays. Gene names and symbols were obtained from Locuslink database. If no name appeared in the Locuslink database, gene name was presented as annotated in the Affymetrix dataset. If EST was displayed on the Affymetrix spreadsheet, a Blast analysis followed by Locuslink search was performed. Both Locuslink and Blast searches were performed at the NCBI website (www.ncbi.nlm.nih.gov/).

2.3 cDNA probes
Probes for heat shock proteins 70 (Hspa1b) and 27 (Hsp27), plasminogen activator inhibitor-1 (Pai1) and S100 calcium binding protein A9 (calgranulin B) were amplified from a rat heart cDNA library (Stratagene, La Jolla, CA, USA). The sequences of the primers and optimal annealing temperatures are presented in Table 1. PCR was carried out with 40 cycles of 1 min at 94°C +1 min at optimal annealing temperature +1 min at 72°C, followed by 5 min at 72°C in the presence of 2.5–5.0 mM of MgCl2. The identities of the probes were confirmed by sequencing, and/or by the size of the transcript on Northern Blot.


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  		Table 1 Primers and clones used to generate northern blot probes

 
2.4 Northern blot hybridization
A 30-µg amount of total RNA isolated from Ctrl and SO hearts and from the NI and Isc tissues as well was subjected to agarose gel electrophoresis and transferred to nylon membrane (Osmonics Westboro, MA, USA). Blots were stained with methylene blue and imaged in the Alpha Imager 2000 (Alpha Innotech, San Leandro, CA, USA). A 50-ng amount of radiolabeled probe was hybridized with the blots followed by autoradiography at –80°C.

The intensities of the spots were scanned using Alpha Imager 2000 and normalized to 18S and 28S RNA values.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Statistical analysis of gene array data
The recommendations from Affymetrix suggest only evaluating transcripts whose ratio between samples was >2.0. As an additional conservative measure we only considered the lower end of the 95% confidence interval for all ratios presented in the text and shown in the tables. To adopt an even more exacting standard, we applied a statistically rigorous model-based analysis of gene expression using the DCHIP software package [14,15]. This results in estimates of ratio and an associated standard error for each gene. DCHIP software explicitly uses data for each probe pair on the chip, rather than using the summary statistics provided in a standard Affymetrix analysis. It has proven successful with numerous data sets [16,17]. Affymetrix produces an image file in which the intensity of the spot corresponding to a given probe is a reflection of the degree to which the probe has hybridized to a transcript. In particular, for a given gene, we define yij, the intensity of the jth probe on ith chip, to be

Formula
where {Theta}i is the expression level for the gene in chip i, {Phi}j reflects the rate at which probe j hybridizes to the gene and eij is the error term [15]. We fit the model to eight datasets in order to identify and remove outlier probes (e.g. probes that are likely to be crosshybridizing, or have other undesirable characteristics). We chose to adopt stringent filtering criteria when identifying up- or down-regulated genes. Mean expression levels are calculated for each gene on each chip, along with associated standard errors. We then select genes that show evidence of having changed expression levels. Such genes are defined to satisfy all of the following criteria: (i) the 95% confidence interval for the ratio lies entirely above +2 for upregulated genes, or below –2 for downregulated genes in all experiments, (ii) the absolute difference in mean expression is at least 100 arbitrary units. These criteria are substantially more stringent than usually employed for Gene Chip analysis, since we work with a confidence interval for the ratio rather than the ratio itself. Our approach is designed to restrict the number of ‘false positives’. While a rather high threshold for inclusion (i.e. difference in the intensities no less than 100 units) will lead to our not including genes with smaller changes, it also allows us to significantly reduce the number of ‘false positive’ genes we might have picked up. At low expression levels, small changes in gene expression could lead to large changes in their ratios. For the aims of our study (i.e. to identify strong and consistent changes in the expression pattern) we feel confident that this approach is both statistically and biologically justified. Altogether we employed eight chips in two hybridization experiments, and analyzed them in a pairwise manner for all chips; i.e. two sets of four independent contrasts were analyzed.

3.2 Gene chip analysis
When the NI tissue was compared to the SO hearts, we found only three marginally downregulated transcripts in the NI tissue. There were no other changes in the gene expression in this comparison that met our conservative criteria. Since the U34 Gene Chip analysis includes 8800 probes, these slight changes in only three transcripts essentially represent no change in mRNA levels in the NI tissue compared to the SO hearts. In striking contrast, however, both the NI tissue and the SO hearts demonstrated much greater changes when compared to the Ctrl hearts. Since no differences were established between the SO hearts and the NI tissue, we combined genes undergoing changes in both SO vs. Ctrl and NI vs. Ctrl comparisons. Only genes demonstrating consistent changes (i.e. altered patterns in both sets of chips) were considered (Table 2). We estimate the ‘false positive’ rate for Table 2 as follows. In this comparison (i.e. SO and NI vs. the Ctrl) we found 24 transcripts. In several instances genes had more than one probe on the chip. After reporting one probe for every gene, we have 18 genes. No downregulated genes were found in these comparisons. In the SO vs. the Ctrl comparison we found 43 upregulated transcripts. In the NI vs. the Ctrl comparison the number of the upregulated transcripts was 25. If the resulting lists were a product of a random noise, we would expect to find 43x25/8800=0.12 transcripts in common, compared with the 24 we actually found. Since this number is significantly smaller than the actual number of identified transcripts (i.e. 0.12 vs. 24) it is likely that genes listed in the Table 2 represent genuine changes rather than chance effects. (This confidence is further reflected by the presence of six duplicate transcripts in the original list of twenty-four transcripts). Changes between the both SO hearts and Ni tissue vs. the Ctrl hearts are presented as means of the ratio's confidence interval lower level from both comparisons; i.e. SO vs. Ctrl and NI vs. Ctrl (Table 2). The majority of altered transcripts (i.e. 11 out of 18) were for inflammation-related genes encoding CCAAT/enhancer binding proteins β and {delta} (Cebpb and Cebpd), Gro (Gro1) calgranulins A and B (S100a8 and S100a9), small inducible cytokine A2 (Scya2), vascular cell adhesion molecule 1 (Vcam1), early growth response 1 (Egr1), zinc finger protein 36 (Zfp36), chemokine CX3C (Cx3cl1), and intercellular adhesion molecule (Icam1).


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  		Table 2 Changes in gene expression patterns in the sham-operated hearts and the nonischemic tissue compared to the control hearts

 
When the Isc tissue was compared with the NI, we found 30 altered transcripts representing 19 genes (Table 3). We estimate the ‘false positive’ rate for this table as follows. The upregulated genes were constructed from 121 and 264 upregulated transcripts found in the 1st and the 2nd sets of chips, respectively. If the resulting list were a product of a random noise, we would expect to find 121x264/8800=3.63 transcripts in common, compared with the 27 we actually found (after reporting one transcript for every gene, we have 16 upregulated genes in this list). The downregulated genes were constructed from lists containing 38 and 144 altered transcripts from the 1st and the 2nd sets of chips, respectively. If the downregulated transcripts represented noise rather than a signal, we would expect to find 38x144/8800=0.62 transcripts in common, compared to the three we actually found. The difference in these numbers (i.e. expected vs. actually found transcripts) indicates that the reported genes are highly likely to be a product of genuine changes rather than of a random noise. The use of conservative criteria for declaring genes to have changed expression allowed us to greatly reduce the number of ‘false positive’ signals.


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  		Table 3 Changes in gene expression pattern in the ischemic vs. the non-ischemic tissue

 
The most highly upregulated gene transcript was for the heat shock protein 70 (Hspa1b) with a ratio +27.6. Ten transcripts were moderately upregulated (ratio +3–5). Among them were transcripts for Hsp27, Hsp105, and Hsp86, small inducible cytokine A2 (Scya2), growth and trophic factors (vascular endothelial growth factor—Vegf, and brain-derived neurotrophic factor—Bdnf), transcription factors (Atf3 and Egr1), serine protease inhibitor-plasminogen activator inhibitor 1 (Pai1) and B-cell translocation gene 2 (Btg2). Five transcripts were marginally upregulated (ratio +2 –3). Among them were mRNAs for DnaJ-like protein (Hsj2 also known as Hsp40), monoamine oxidase A (Maoa), growth arrest and DNA damage inducible 45 {alpha} (Gadd45a), L1 transposone and transcript similar to filamin C. Two genes underwent marginal downregulation (ratios –2.9 and –3.9, respectively) and one transcript was significantly downregulated (ratio –15.9). The latter was represented by the atrial myosin light chain-1 (AMLC-1) (Table 3).

3.3 Northern blot analysis
We used Northern Blot analysis to confirm selected results obtained with Affymetrix gene chip microarray. Hybridization patterns and 28S and 18S RNA staining are presented in Fig. 1. Intensities recorded by the Affymetrix method and signals on Northern blots normalized to 18S and 28S RNAs are presented in Fig. 2. Northern blot results indicate that transcripts for Hsp70 (Hspa1b), and Hsp27 were highly upregulated in the Isc tissue. Transcripts for calgranulins A and B (S100a8 and S100 a9) were upregulated in the SO hearts and the NI and Isc tissues compared to the Ctrl hearts. Pai1 (plasminogen activator inhibitor 1) expression was increased in the SO hearts when compared to the Ctrl hearts, and also in the Isc area compared to the Ctrl and NI tissues. Fig. 2 also indicates that the patterns of the intensities were similar on both the gene chip array and on the Northern blots.


Figure 1
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  		Fig. 1 Northern blots for Hsp70 and 27, calgranulins A and B, and plasminogen activator inhibitor-1.

 

Figure 2
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  		Fig. 2 Comparison of gene array and Northern blot results for Hsp70 and 27, calgranulins A and B, and plasminogen activator inhibitor-1. Bars represent intensities recorded from Gene Chips (left panel) and normalized to 18S and 28S RNAs intensities from Northern blots (right panel).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Our results indicate that brief coronary artery ischemia of 20-min duration induced changes in the pattern of mRNA expression in the affected myocardium that were detected 4 h following the ischemic insult. In addition, mere exposure to a surgical procedure not necessarily involving the heart (as indicated by changes in the SO and NI vs. the Ctrl hearts of animals not operated upon) also altered the levels of a number of mRNAs.

A single brief episode of ischemia caused a strong upregulation of genes encoding heat shock proteins 70 (Hspa1b), 27 (Hsp27), 105 (Hsp105), 86 (Hsp86) and 40 (Hsj2) in the Isc tissue when compared with the NI area (Table 3). Experiments with heat shock proteins induction with hyperthermia, their overexpression in transgenic mice and direct transfer into the myocardium as well showed that these proteins could render the heart resistant to an ischemic insult [18–20].

We also observed increases in growth factors (Bdnf and Vegf), activating transcription factor 3 (Atf3), plasminogen activator inhibitor 1(Pai1), B-cell translocation gene 2 (Btg2), and growth arrest and DNA damage inducible 45 {alpha} (Gadd45a) mRNAs. Growth factors by their designation are cell growth promoters and can be considered protective. Upregulation of Bdnf may represent a response aimed at preventing neuronal injury in the ischemic heart. Stimulation of Vegf was shown to increase vascularization in ischemic tissue [5]. Atf3, a transcription factor also known as liver regenerating factor, was suggested to play an important role in growth regulation by controlling the expression of delayed genes [21]. Recently it was shown that Atf3 inhibits doxorubicin-induced apoptosis in cardiac myocytes [22]. Our studies may indicate a possible protective role for Atf3 in damaged tissue. Pai1 (a serine protease inhibitor with antiapoptotic properties), Btg2 (a cell survival promoter), and Gadd45a (controlling DNA repair, genomic stability and cell resistance to stress) also could be considered as protective factors [23–26].

Increases in inflammation-related transcripts in the SO and the NI tissues (i.e. Cebpb, Cebpd, Gro1, S100a8, S100a9, Scya2, Vcam1, Egr1, Zfp36 and Cx3c11) most likely represent a general response to injury (Table 2). Calgranulins A and B, Scya2 and Egr1 are induced in the Isc tissue as well (Tables 2 and 3Go, Figs. 1 and 2Go). These proteins are closely involved in inflammatory processes, and thus their increase in all three tissues subjected to surgical trauma is not surprising. Some of the transcripts upregulated in response to surgical injury require a coordinated interaction; e.g. chemokines Cx3cl1 and Gro1 both mediate monocyte arrest via Cx3cl1 receptor and calgranulins A and B (S100a8 and S100a9) act as heterodimer [27,28]. Significant downregulation of the AMLC-1 transcript in the ischemic area (Table 3) may represent an ischemia-related injury in the atrium since the left coronary artery in the rats supplies atrial tissue.

Since the U34 gene chip analysis includes 8800 probes, changes in only three transcripts in the NI vs. SO hearts were considered as no change in mRNA levels in these tissues. However, when less stringent criteria were applied (i.e. lower level of the ratio's confidence interval +1.5 for upregulated and –1.5 for downregulated mRNAs) we found five increased and twenty-five decreased transcripts in the NI compared to the SO tissues. These changes in mRNA levels indicate certain differences in the response generated in the remote NI tissue from the heart subjected to regional ischemia, compared to the SO hearts. NI tissue must sustain the increased hemodynamic demand of the noncontractile ischemic tissue; while SO hearts obviously do not experience this increased hemodynamic stress.

The upregulation of specific mRNAs only in the Isc tissue indicates that genetic reprogramming induced by a brief episode of ischemia can be clearly distinguished from changes in gene expression pattern caused by surgical trauma per se. Such transcripts included those for protective heat shock proteins, growth and trophic factors, inhibitors of apoptosis, transcription factors and survival promoting molecules; i.e. Hsp70 (Hspa1b), Hsp27, Hsp105, Hsp86, Hsj2, Vegf, Bdnf, Atf3, Pai1, Btg2, and Gadd45a. The cardioprotective role of heat shock proteins is a well-established fact [18,19]. In addition, early changes in antiapoptotic and trophic genes could also play a significant role in cardioprotective response shortly after brief episodes of ischemia. However, the long term input of observed changes in terms of their effect(s) upon the expression of critical proteins, needs to be addressed. In sharp contrast to this early ‘protective’ response to brief nonlethal ischemia, late changes in gene expression patterns following myocardial infarction (as indicated by gene chip studies in experimental models of lethal ischemia) are represented by upregulation of hypertrophy- and remodeling-related transcripts which are major contributors to both structural damages and functional decline as well in the postinfarcted myocardium [7–10].

Some of the changes observed in our study confirm previously known observations. Heat shock protein 71 was shown to be increased in the isolated perfused rat heart after 1 h of ischemia, mRNA for Scya2 (JE/MCP-1 gene) was increased in the in vivo mouse heart ischemia model following 1 h of permanent coronary artery occlussion, Atf3 mRNA was induced in the in vivo rat heart ischemia model following both 2 h of ischemia alone or combined with 1 h of reperfusion as well, Egr1 mRNA was upregulated with 10 min of regional ischemia in the in vivo swine heart ischemia model, circulating Vegf protein was increased gradually in patients after the onset of acute myocardial infarction, and both Gadd45a mRNA and protein as well were increased in the rat brain after 4 h of focal ischemia [29–34]. These studies utilized different experimental models of ischemia, included clinical settings, and different species and tissues as well. The use of gene chip technology enables investigators to perform a global gene profiling, and to summarize organ and tissue specific changes in the expression patterns occurring within different categories of genes and to attribute them to a specific condition and/or treatment. This approach demonstrated activation of a protective or ‘survival’ program in a swine ischemia model [35]. The authors confirmed a strong upregulation for Hsp70, Egr1, Pai1 and Pai2 in the ischemic area. Our study confirms the existence of a related survival program in rats. In addition to known factors (heat shock proteins, Scya2, Atf3, Egr1, Vegf and Pai1) we were able to detect several other transcripts i.e. Bdnf, Btg2 and Gadd45a upregulated only in the ischemic myocardium. However, the timing of this program and the specific genes undergoing activation may differ depending on the species and the model employed. For example, our previous study utilizing rabbit ischemia model and gene macro- (rather than micro-) array indicated that brief episodes of ischemia caused a strong upregulation in a transcript encoding protective mitogen activated protein kinase activated protein kinase (i.e. MAPKAPK 3) [36].

In conclusion, the use of the gene microarray technology allowed us to analyze a broad array of data and to characterize the observed changes as a protective genetic program activated in the rat heart, when it is subjected to a single brief episode of ischemia. This early genetic program represents a legitimate target for elucidating the mechanisms that promote the protective effect and reduce the disease-related changes in the pattern of gene expression.

Time for primary review 21 days.


   Acknowledgements
 
This work was supported in part by NIH grant 5RO1-HL52771-07 (LHK, RAK) grants from the Greater Los Angeles American Heart Association (LHK, RAK), grant-in-aid from the American Heart Association Western States Affiliate (CP), The Kenneth T and Eileen L. Norris Foundation and The Joseph Drown Foundation (RAK). We thank Joan Dow for surgical preparation.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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