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Cardiovascular Research 2003 58(1):126-135; doi:10.1016/S0008-6363(02)00845-3
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Copyright © 2003, European Society of Cardiology

Characterization of pDJA1, a cardiac-specific chaperone found by genomic profiling of the post-ischemic swine heart

Christophe Deprea,b,*, Li Wanga,b, James E. Tomlinsonc, Vinciane Gaussina,b, Maha Abdellatifa,b, James N. Topperc and Stephen F. Vatnera,b

aDepartment of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Street, MSB G-609, Newark, NJ 07103, USA
bDepartment of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA
cCOR Therapeutics Inc., South San Francisco, CA, USA

* Corresponding author. Tel.: +1-973-972-3926; fax: +1-973-972-7489. deprech{at}umdnj.edu

Received 16 August 2002; accepted 4 December 2002


   Abstract
Top
 Abstract
1 Methods
 2 Results
 3 Discussion
 References
 
Background: Previously, we showed by subtractive hybridization in a swine model of ischemia/reperfusion that an upregulation of genes participating in mechanisms of cell survival is a potential genomic mechanism to tilt the balance from necrosis to functional reversibility. Methods and results: We present here the full-length sequencing and characterization of a novel gene that was found in this subtraction, encoding a cardiac-specific DnaJ-like co-chaperone that we call Pig DnaJ-like protein A1 (pDJA1). The expression of pDJA1 was found to be restricted to the heart, as opposed to skeletal muscle, liver, lung, kidney, aorta, stomach and spleen. Expression of pDJA1 is restricted to cardiac myocytes, as determined by in situ hybridization. The transcript is expressed more in the left ventricle than in the other cardiac chambers. Remarkably, expression of pDJA1 follows a transmural gradient in the left ventricle, with the highest level of expression in the subendocardium. Expression of pDJA1 slightly increased during an episode of ischemia, but increased by 4-fold during the following period of reperfusion. Adenovirus-mediated transduction of pDJA1 in isolated rat neonatal cardiac myocytes decreased by 65% the rate of apoptosis induced by staurosporine. Conclusion: Therefore, pDJA1 is a novel heart-specific, ventricle-enriched cardioprotective co-chaperone, which participates in the program of cell survival that limits irreversible damage in post-ischemic myocardium.

KEYWORDS Gene expression; Ischemia; Stunning

Heat shock proteins are involved in the folding, degradation and translocation of intracellular proteins [1], but they also participate in the protection against apoptosis and in cell growth [2–5]. They are crucial effectors of the program of cell survival, which protects cells against irreversible damage and accelerates functional recovery after stress [6]. Two main forms of heat-shock proteins in E. coli, called DnaK and DnaJ, have been conserved in eukaryotes [7]. In mammalian cells, the chaperone HSP40 is the homologue of DnaJ. Several isoforms of DnaJ-like/HSP40 homologues have been cloned, that differ by their tissue distribution and their protein interactions. The role of these co-chaperones is to stimulate the ATPase activity of the cognate HSP70 [8,9] and to modulate its substrate-binding capacity. The heat-shock response is particularly developed in cardiac cells, which are long-lived, post-mitotic cells submitted to high oxidative stress [10]. During ischemia/reperfusion, this response is important to tilt the balance between cell survival and cell death

Myocardial stunning refers to a form of non-lethal, fully reversible myocardial dysfunction that follows an acute episode of ischemia [11,12]. The syndrome of stunning is prevalent in different etiologies of coronary artery disease, including stable or unstable angina pectoris, myocardial infarction, and post-surgical dysfunction [13]. Due to the major prevalence of ischemic heart disease, stunning is of paramount importance because it corresponds to a condition in which myocardial viability is maintained. Unraveling the molecular mechanisms of cardioprotection in stunned myocardium can open new avenues to salvage dysfunctional cardiac tissue and prevent cardiac cell loss. Especially, a better understanding of the mechanisms by which the molecular and cellular adaptations maintain cell survival should open new therapeutic opportunities.

Recently, we showed by cDNA subtractive hybridization the array of genes that are upregulated by ischemia–reperfusion in a large mammalian model of myocardial stunning [14]. Specifically, half of the genes that were upregulated in ischemic myocardium encode transcripts that are involved in protective mechanisms against irreversible ischemic damage, including heat-shock proteins [14]. We therefore proposed that ischemia–reperfusion triggers the expression of a program of cardiac cell survival, which may limit cell necrosis and apoptosis. Because of the large-scale, unbiased approach of the subtractive hybridization, some of the products that were sequenced did not recall any product described in public databases. We present here the full-length sequencing and characterization of one of these genes, which we identified as a cardiac-specific DnaJ-like co-chaperone. This transcript is characterized by a remarkable tissue distribution and by a strong upregulation during ischemia–reperfusion. In accordance with a proposed classification of HSP40 homologues [15], we call this transcript pDJA1, for pig DnaJ-like protein A1. Upregulation of pDJA1 during reperfusion further expands the concept of a program for cell survival that prevents irreversible damage in post-ischemic myocardium.


   1 Methods
Top
 Abstract
 1 Methods
2 Results
 3 Discussion
 References
 
1.1 Instrumentation
Female domestic swine (22–25 kg) were anesthetized with thiopental sodium (5–10 mg/kg, i.v.) and isoflurane (0.5–1.5 vol%). A left thoracotomy was performed through the fifth intercostal space to expose the heart [16]. A hydraulic occluder was implanted around the base of the left anterior descending (LAD) artery. Myocardial blood flow through the LAD was monitored by a Doppler flow probe. After 3 days of recovery, stunning was induced in the conscious animal by inflating the coronary occluder, to reduce the blood flow in the LAD by 40%. Reduction of the blood flow was controlled on-line via the flow probe. The coronary stenosis was maintained for 90 min, followed by deflation of the occluder and full reperfusion. Animals were anesthetized at the end of the 90-min stenosis period (n = 5), or after 1 h (n = 5) and 12 h (n = 5) reperfusion. In each case, myocardial samples were taken from both the stunned area (centrally in the LAD territory) and the remote area of the beating heart. Each sample was further separated in a subendocardial and a subepicardial portion. Three instrumented pigs, in which no occlusion was performed, were used as shams. Samples from both atria and from different organs (kidney, liver, lung, spleen, aorta, skeletal muscle, stomach) were taken as well. The samples were frozen in liquid nitrogen or fixed in fresh 4% paraformaldehyde. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

1.2 RNA extraction
About 300 mg of each sample were homogenized in 3 ml of the guanidium thiocyanate–phenol–chloroform solution (Triazol, Gibco Life Technologies). Total RNA was extracted [17], resuspended in 50 µl DEPC–water, and its concentration was measured spectrophotometrically by the absorbance at 260 nm. The integrity of the RNA pool was checked on a 1% agarose denaturing gel stained with ethidium bromide.

1.3 Cloning of PDJA1
A pig heart cDNA library was subcloned in the pCMV Sport6 vector (Life Technologies) and used for screening with primers designed from the sequence obtained in the subtractive hybridization. The cDNA was obtained by PCR cloning and colony hybridization. The 5'-end of the transcript was obtained by 5'-RACE after decapping of the transcripts (First Choice RLM-RACE, Ambion). PCR products were sequenced by triple pass on a 3100 Genetic Analyzer (Applied Biosystems) using the Big-Dye Terminator (Applied Biosystems). Data analysis was performed with the ABI AutoAssembler software. Gene analysis and sequence comparisons were performed with the MacVector software.

1.4 Northern blotting
Fifteen micrograms of total RNA were applied on a 1.2% agarose denaturing gel stained with ethidium bromide. After migration, the RNA was transferred overnight to a nylon Hybond-N membrane (Amersham–Pharmacia), then cross-linked by UV. A probe was derived as an isolated restriction fragment from the subtractive library, heat-denatured, and labeled with [{alpha}-32P]dCTP (Prime-It II kit, Stratagene). Hybridization was performed overnight at 42°C in a hybridization solution containing 50% formamide. Intensity of the radioactive signal was measured with the Multi-Analyst detection system (Biorad). RNA integrity was controlled by comparison of the bands corresponding to the 28S and 18S rRNAs.

1.5 Quantitative RT-PCR
Expression of pDJA1 was measured by quantitative RT-PCR on a 7700 Sequence Detector (Applied Biosystems) with specific primers (forward: 5'-CTCTCTTGGAAGCTTCCTGAAC-3', reverse: 5'-GCACTGCAAAGGCTGTCAA-3') and a fluorescent probe (5'-FAM-AAGCTTGTGGTGAGGACAAACCAGTGTTT-3' TAMRA). The mRNA of interest was reverse-transcribed from 60 ng of total RNA, and subsequently used for quantitative two-step PCR (40 cycles of a 10-s step at 95°C and a 1-min step at 60°C). Internal RNA standards were prepared from the PCR-amplified cDNA after ligation of the T7 promoter using the MegaShortScript kit (Ambion, Austin, TX) [18]. The values of the transcript were normalized to the transcript level of cyclophilin, measured in each sample as an internal control.

1.6 In situ hybridization
Samples were fixed in 4% paraformaldehyde–PBS, embedded in paraffin and sectioned at 6-µm intervals. Sections were dewaxed, rehydrated in ethanol, and treated with 0.8% pepsin in 0.2 N HCl (Dako) for 5 min at 37°C, followed by a 5-min rinse in H2O. Sections were then re-fixed for 20 min in 4% paraformaldehyde dissolved in PBS. After washing, sections were acetylated in 0.25% acetic anhydride diluted in 0.1 M triethanolamine buffer (pH 8.0). Sections were hybridized overnight at 37°C in a humidified chamber with a biotin-labeled oligonucleotide probe diluted in hybridization solution (Dako), corresponding to the same probe as the one used for the quantitative PCR. Probe hybridization was detected with streptavidin–alkaline phosphatase, after addition of BCIP/NBT as a chromogenic substrate (Dako).

1.7 Adenovirus-mediated transfer of pDJA1
Primary cultures of ventricular cardiac myocytes were prepared from 1-day-old Wistar rats. Cardiac myocytes were dispersed from the ventricles by digestion with 0.1% collagenase type IV (Worthington), 0.1% trypsin (Gibco) and 15 µg/ml DNase I (Sigma). Cell suspensions were applied on a discontinuous Percoll gradient (1.060/1.082 g/ml) made up in DF buffer containing Dulbecco's modified Eagle medium (DMEM)–F12 (1:1, Invitrogen), 17 mM NaHCO3, 2 mM glutamine and 50 µg/ml gentamycin. Cardiac myocytes were plated on culture dishes at a density of 106 cells per well. The culture medium was changed to a serum-free medium after 24 h.

The coding sequence of pDJA1 was ligated downstream of the CMV promoter in a pDC315 shuttle vector. An adenovirus harboring LacZ was used as a negative control. The recombinant adenoviruses (Ade-pDJA1 and Ade-βGal) were then prepared in 293 cells by cotransfection of a cosmid containing the adenovirus type 5 genome (devoid of E1 and E3) with the shuttle vector, using lipofectamine (Gibco). Titers were determined on 293 cells overlaid with DMEM plus 5% equine serum and 0.5% agarose. After 24 h in culture, cardiac myocytes were infected in serum-free medium with the Ade-pDJA1 or the AdeβGal adenovirus. Twenty-four hours after infection, apoptosis was induced by addition of 4 µM staurosporine (Sigma) dissolved in DMSO, and quantified by the activation of caspase-3 (ApoTarget, BioSource).

1.8 Statistical analysis
Data are expressed as mean±standard deviation. The number of samples in each experiment is indicated in the figure legends. Statistical analysis was performed with the Student's t-test. A value of P<0.05 was considered as significant.


   2 Results
Top
 Abstract
 1 Methods
 2 Results
3 Discussion
 References
 
2.1 Cloning of pDJA1
In addition to the known genes that were found in the subtractive hybridization between stunned and normal pig myocardium [14], a 0.8-kb cDNA fragment was subcloned which did not match any known transcript in public databases. To determine the full-length sequence of this unknown transcript, we screened a pig heart library with primers designed from the 0.8-kb fragment, and amplified the products by PCR. With this method, 685 nucleotides of the 3'-end including a poly-adenylation signal and the poly-A tail were obtained. Next, we used 5'-RACE PCR to obtain the remaining 5'-end portion of the transcript. Taken together, a full-length transcript corresponding to a 3.1-kb long cDNA was obtained (Fig. 1), which is characterized by a 62-nucleotide long 5'-UTR, a 397 amino acid open reading frame and a 1.75-kb long 3'-UTR. The open reading frame begins with the ATG at nucleotide 63, and is not preceded by a Kozak's consensus for translation initiation [19]. The protein has an apparent molecular weight of 44.7 kDa and a pI = 8.27. The protein contains the N-terminal J domain characteristic of the DnaJ-like/HSP40 homologues, followed by a glycine-rich stretch and four ‘zinc finger’ CxxCxGxG motifs. Interestingly, the C-terminus contains a CaaX prenylation site, which usually characterizes proteins involved in cell growth. The long 3'-UTR contains seven AU-rich mRNA decay elements [20], characterized by the sequence AUUUA. This sequence interacts with RNA-binding proteins, which regulate the stability and half-life of transcripts usually encoding proto-oncogenes and cytokines [21]. The 3'-UTR ends with a poly-adenylation signal at nucleotide 2980. Fig. 2 shows the protein sequence alignment between pDJA1 and the human HSP40. Both proteins share a homologous N-terminus, which includes the J domain and the G/F tract. pDJA1 totally diverges from HSP40 in its C-terminal part, including the prenylation site which is absent in HSP40 (Fig. 2).


Figure 1
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  		Fig. 1 Nucleotide and amino acid sequence of pDJA1. The figure shows the full-length transcript encoding pDJA1. The N-terminal J domain and the C-terminal prenylation site are boxed. The four zinc fingers motifs are underlined with a solid line. The glycine-phenylalanine stretch is underlined with a dotted line. The AU-rich motifs in the 3'-UTR are shadowed. The poly-A signal is underlined with an arrow.

 

Figure 2
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  		Fig. 2 Protein sequence alignment between the prototypic human HSP40 (acc. BC002352) and pDJA1. The alignment shows that both molecules are highly similar in the N-terminal part, which includes the J domain and the G/F tract. PDJA1 markedly diverges from, and is longer than, HSP40 in its C-terminal part.

 
2.2 Tissue distribution of pDJA1
A pig multi-tissue Northern blot was probed, using the original 0.8-kb fragment of the subtractive hybridization (Fig. 3). This Northern blot showed one specific band at 3.1 kb, corresponding to the full-length transcript. Remarkably, the expression of pDJA1 was specific for the heart, as it was not detected in the other pig tissues tested, such as stomach, kidney, liver, lung, spleen, aorta or skeletal muscle. The distribution of the pDJA1 transcript in myocardial tissue under baseline conditions was further investigated and compared to the expression of other heat-shock proteins by quantitative PCR (Fig. 4). As shown in Fig. 4A, a higher level of expression of pDJA1 was found in the ventricles when compared to the atria, but the expression in the left ventricle was 2-fold higher than in the right ventricle. Interestingly, a separate analysis of subendocardial and subepicardial samples from the left ventricle showed that the expression of the pDJA1 transcript was double in subendocardium over subepicardium (Fig. 4B). This distribution is specific of pDJA1, because the transcript level of other heat-shock proteins highly expressed in the heart, such as HSP70 and HSP40, did not show any gradient of expression in normal left ventricle (Fig. 4C).


Figure 3
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  		Fig. 3 Tissue distribution of pDJA1 in the swine. Northern blot performed on different pig tissues with a probe corresponding to a 0.8-kb fragment of pDJA1 found in the subtractive hybridization. Ribosomal RNAs (28S and 18S) are shown for equal loading.

 

Figure 4
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  		Fig. 4 Characterization by quantitative PCR of the expression of pDJA1 compared to other heat-shock proteins in the normal swine heart. (A) The measurement of the pDJA1 transcript by quantitative PCR in the different cardiac chambers (n = 4 per group). **P<0.01 versus both ventricles; *P<0.05 versus left ventricle. (B) The different expression of pDJA1 between sub-endocardium (sub-endo) and sub-epicardium (sub-epi) in left ventricle (n = 4 per group). *P<0.05 versus sub-endocardium. (C) The expression of other heat-shock proteins, such as HSP70 and HSP40, does not differ transmurally in normal left ventricle (n = 4 per group). Cyclophilin mRNA was used as a normalizer.

 
2.3 Upregulation of pDJA1 transcript during ischemia/reperfusion
The pDJA1 transcript was found in the subtractive library of stunned myocardium, suggesting that this transcript is upregulated by ischemia. To confirm this, four pig hearts were submitted to 90-min coronary stenosis, followed by 1-h reperfusion. The expression of pDJA1 in the ischemic area and remote area of the same hearts was measured by Northern blot, and the signal was normalized to the band of the 28S ribosomal RNA. As shown in Fig. 5A, the expression of pDJA1 was increased about 4-fold in the reperfused myocardium.


Figure 5
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  		Fig. 5 Upregulation of pDJA1 gene expression by ischemia–reperfusion. (A) The expression of pDJA1 measured by Northern blot in left ventricular samples from four hearts submitted to regional ischemia for 90 min, followed by 1 h reperfusion. In each case, a sample from the remote area and from the ischemic area were measured in parallel. Normalization of the pDJA1 signal to the 28S rRNA signal showed that pDJA1 was increased 4-fold in stunned myocardium compared to remote area. *P<0.05 versus remote. (B) The quantitative measurement of the pDJA1 transcript in samples of the remote (open symbols) and stunned area (closed symbols), from hearts submitted to 90 min occlusion followed by no reperfusion, 1 or 12 h reperfusion (n = 5 in each group). The subendocardial (circles) and subepicardial areas (squares) were measured separately. *P<0.05 versus corresponding value in remote myocardium. (C) In situ hybridization (magnification, x40) showing that the expression of the pDJA1 gene in stunned myocardium is myocyte-specific.

 
To further determine the time-course of this increased expression, additional animals were sacrificed at the end of the 90-min occlusion period, or after 12-h reperfusion. Sham-operated animals, in which no coronary stenosis was performed, were also included to test the stability of the remote area throughout the protocol. As shown on Fig. 5B, the level of the pDJA1 transcript slightly increased in the subendocardium during the ischemic episode. However, a maximal and transmural increase was observed at 1-h reperfusion. The difference of expression between subendocardium and subepicardium found in control hearts persisted at all time-points during stunning. At 12 h reperfusion, the pDJA1 transcript returned to normal values in the ischemic tissue (Fig. 5B). This time-course is similar to that observed for most of the genes which are upregulated in this model of stunning and parallels the progressive functional recovery of stunned myocardium [14]. The level of the pDJA1 transcript in the remote area was similar to that in sham animals at all time-points. We determined that this increase in pDJA1 expression was myocyte-specific by in situ hybridization. As shown in Fig. 5C, a strong expression was found in cardiac myocytes from ischemic myocardium, whereas a faint signal was detected in normal myocardium. No signal was detected in endothelial cells.

2.4 Cytoprotective effect of pDJA1 in isolated cardiac myocytes
To confirm that pDJA1 is a co-chaperone participating in cell survival, isolated cardiac myocytes were infected with an adenovirus containing the coding sequence of pDJA1 under the control of the CMV promoter (Ade-pDJA1), and compared with an adenovirus harboring an irrelevant sequence (Ade-βGal vector). Programmed cell death (apoptosis) was induced by addition of 4 µM staurosporine for 1 h and quantified by the measurement of caspase-3 activation. Fig. 6 shows the increase of apoptotic rate in presence of staurosporine as a percentage of the value found in both groups in absence of staurosporine. After addition of staurosporine, the stimulation of apoptosis in cells transduced with pDJA1 was 65% lower than the values observed in the cells transduced with the control Ade-βGal vector (Fig. 6). Therefore, these data in vitro confirm that overexpression of pDJA1 in myocardium confers a cytoprotective effect.


Figure 6
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  		Fig. 6 Cytoprotective effect of pDJA1 in isolated cardiac myocytes. Isolated cardiac myocytes were infected with an adenovirus containing the coding sequence of pDJA1 (Ade-pDJA1), and compared with an adenovirus harboring an irrelevant sequence (Ade-βGal vector). Apoptosis was induced by addition of 4 µM staurosporine for 1 h and quantified by the measurement of caspase-3 activation (n = 3). Values are expressed as the increase in apoptotic rate in both groups after addition of staurosporine (–staur. versus +staur.) *P<0.05 versus corresponding value in Ade-βGal group.

 

   3 Discussion
Top
 Abstract
 1 Methods
 2 Results
 3 Discussion
References
 
3.1 Structure of pDJA1
We report the full-length cloning and characterization of pDJA1, an HSP40 homologue which is specifically expressed in the heart. The DnaJ homologues belong to a family of proteins regulating the activity of HSP70 [8,9] and HSP90 [22]. Their characteristic motifs confer the HSP40 homologues a co-chaperone function, by which they interact with other heat-shock proteins and target them to a specific intracellular localization [23,24]. Specifically, the DnaJ homologues are particularly important to couple the ATPase and substrate-binding properties of HSP70, to ensure optimal coupling between ATP hydrolysis and protein refolding [8,9,25]. pDJA1 contains the canonical motifs of this protein family, including a J domain, a glycine–phenylalanine tract and four zinc finger motifs. While DnaJ homologues bind to HSP70 by the J domain [7,26], they tether the unfolded proteins in their zinc finger motifs [27], using the glycine–phenylalanine tract as a hinge to assure contact between both proteins.

3.2 Role of heat-shock proteins during ischemia–reperfusion
Ischemia–reperfusion upregulates the expression of heat-shock proteins, which participate in the protection of myocardium against irreversible ischemic damage both in vitro and in vivo [28–32]. The results of our experiments in isolated cardiac myocytes illustrate that pDJA1 participates in this cytoprotective mechanism. The mechanisms by which HSPs protect the ischemic heart are diverse. In addition to their classical role in the refolding of denatured proteins [6], each HSP has more specific functions. For instance, HSP90 stimulates the activity of endothelial NO synthase [33], HSP60 and HSP10 protect mitochondrial oxidative function [34], whereas {alpha}B-crystallin stabilizes sarcomeric proteins [35]. Growing evidence shows that heat-shock proteins are also involved in other survival mechanisms, including resistance to apoptosis [2–5] and cell growth [36,37]. Previously, we showed that stunning induces the expression of a program of cell survival, characterized by the upregulation of genes conferring resistance to apoptosis [14]. Included in this program was the upregulation of different heat-shock proteins, such as HSP 70, HSP 28 and {alpha}B-crystallin [14]. These proteins can block specific pathways leading to apoptosis, such as the release of cytochrome c (by HSP60 and HSP27) [34], the activation of Apaf-1 (by HSP90) [38], the activation of caspase-9 (by HSP70) [3] or the activation of caspase-3 (by {alpha}B-crystallin) [5]. The cytoprotective action of HSP70 requires its interaction with HSP40 homologues [39], such as pDJA1. In addition, the carboxy-terminal prenylation site of pDJA1 may involve this protein in the regulation of growth-related pathways, especially the Ras pathway [40]. Such prenylation site is also found in a yeast DnaJ protein, YDJ1p, and is crucial to the function of this molecule [41]. Because the C-terminal part of pDJA1 and HSP40 differ markedly (Fig. 2), these two proteins may interact with HSP70 in different sub-cellular compartments. Therefore, post-ischemic activation of genes encoding novel chaperones reinforces the concept of a genomic program for cell survival, growth and resistance to apoptosis during non-lethal ischemia.

3.3 Regulation of pDJA1 gene expression
The originality of pDJA1 expression in the swine heart can be summarized in three ways: the transcript is specifically expressed in cardiomyocytes, its concentration rapidly increases in response to ischemia/reperfusion, and it shows a transmural gradient of expression even in the normal myocardium.

We show by Northern blot and in situ hybridization that the expression of the gene encoding pDJA1 is characterized by a remarkable cardiac specificity, which is unusual for the family of DnaJ-like HSP40 homologues. Although the expression of specific heat-shock proteins has already been shown to be restricted to skeletal muscle [42], the present study is novel in that it reports the expression of a cardiac-specific co-chaperone. This selective expression of PDJA1 in the heart also implies that the promoter of this gene contains cis-elements recognized by cardiac-specific transcription factors, which remain to be investigated. We cannot totally exclude that pDJA1 could be induced in other tissues under stress. However, pDJA1 is already expressed in normal myocardium, and is further increased after ischemic stress. It is not an ‘all-or-nothing’ induction of the transcript in stress condition. Therefore, considering that the transcript is not expressed at all in other tissues, its induction upon stress in these tissues is less likely.

The pDJA1 transcript is rapidly increased by ischemia and reperfusion (3–5-fold), and its profile of expression correlates with the time-course of functional recovery. Transcription of genes encoding heat-shock proteins can be increased by stretch [43,44], by the increased activity of the hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) [10], and by the production of free radicals associated with reperfusion [45]. All these factors are probably combined in the present experiments, which may explain why pDJA1 starts to increase during ischemia, then maximally increases at reperfusion when free radicals are released. Although the reason for a cardiac-specific expression of pDJA1 probably relies in its promoter, the rapidity of the increase in pDJA1 transcript together with the structure of the 3'-UTR suggest that post-transcriptional mechanisms of mRNA stabilization participate in the increased concentration of the transcript in post-ischemic myocardium. This might be a specific mechanism of regulation, as the prototypic human HSP40 (acc. BC002352 [GenBank] ) has a shorter 3'-UTR and does not display the AU-rich elements found in pDJA1.

Most intriguingly, the transcript is preferentially expressed in the left ventricle, where its pattern of expression follows a transmural gradient, which is quite unusual in normal left ventricle. In a previous study, we showed that most of the genes that are upregulated by ischemia–reperfusion present such a gradient in the ischemic myocardium, which led to the conclusion that the genomic response of the myocardium to ischemia is not an ‘all or nothing’ phenomenon, but is proportional to the severity of ischemia [14]. However, in that study, all the transcripts measured separately in subendocardium and subepicardium of the remote, normoxic myocardium showed a similar level of expression [14]. The pDJA1 transcript is therefore an exception, which suggests that the expression of this gene is exquisitely sensitive to the workload and/or the oxygen concentration. Sensitive oxygen-sensing and/or stretch-sensing mechanisms could explain the higher expression of pDJA1 in the subendocardium. It remains to be determined whether the AU-rich units found in the 3'-UTR are responsible for this regulation. Once an antibody is made available, future experiments will also determine whether the protein expression of pDJA1 correlates with the gene regulation.

In conclusion, we show that pDJA1 is a novel co-chaperone expressed specifically in myocardium, and which is upregulated by ischemia/reperfusion. Experiments conducted in vitro in isolated cardiac myocytes confirm that overexpression of this co-chaperone reduces irreversible damage. The characterization of this transcript extends the concept of a program for cardiac cell survival which is activated in ischemic myocardium [14]. The variation of expression of this transcript in both normoxic and post-ischemic myocardium illustrates original mechanisms of transcriptional regulation in the heart. In addition, this study shows the usefulness of the subtractive hybridization methodology to characterize ‘unexpected’ and novel genes, which may offer a better understanding of the mechanisms of cardiac resistance to ischemia and potential new therapeutic perspectives.

Time for primary review 25 days.


   Acknowledgements
 
This work was supported by National Institute of Health grants HL33065, PO1 HL 59139, AG 14121 and HL 33107 to S.F.V., and AHA Scientist Development Grant 0230017N to C.D.


   References
Top
 Abstract
 1 Methods
 2 Results
 3 Discussion
 References
 

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