Heart transplantation changes the expression of distinct gene families

BEDEL BIYIHA NGIMBOUS1, FRANCINE BOURGEOIS2, CHRISTOPHE MAS2, MICHEL SIMONNEAU2 and JEAN-MARIE MOALIC1

1 Unité 127, Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Lariboisière, 75475 Paris Cedex
2 Neurogénétique/INSERM E9935, Hôpital Robert Debré, 75019 Paris, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We took advantage of the combination of a rat heart transplantation model with a modified differential display RT-PCR method to identify transcriptome changes in the right atria from transplanted compared with native hearts. Based on sequence homology search, the 37 cDNAs differentially displayed both 2 and 7 days posttransplantation were categorized into 7 unknown transcripts, 16 expressed sequence tags (ESTs), and 14 partially or completely characterized genes. The last group cDNAs, validated by relative RT-PCR, belonged to diverse gene families involved in specific metabolisms, protein synthesis, cell signaling, and transcription. Furthermore, we identified differential transcripts corresponding to denervation and fetal gene reexpression. We found coordinate downregulation of genes involved in energy metabolism and protein synthesis regulation, similar to that reported for senescent skeletal muscle. From these transcriptome changes, we propose that heart transplants and senescent muscles share common molecular mechanisms.

heart transplantation; differential display; cardiac denervation; expression profile homologies


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MANY STUDIES HAVE INVESTIGATED the unique physiological features of the transplanted heart, which include alterations in resting hemodynamic function and resting heart rate, blunted heart rate responses to stress and exercise, and distinctive responses to cardiovascular drugs (8). Several hypotheses, some conflicting, have been put forward to explain these physiological alterations and their potential connections with the development of transplant atherosclerosis and rejection (8). The diversity of these hypotheses reflects the restrictions that limit studies in human heart transplant recipients (52) and the paucity of available experimental data on the molecular consequences of heart transplantation. Another stumbling block to the elucidation of heart transplant physiology is the huge complexity of the mechanisms potentially involved: several interacting mechanical and neurohormonal factors, both endogenous and systemic, shape the unique phenotype of the transplanted heart via molecular mechanisms that are largely unknown.

Heart transplantation in the rat offers a unique opportunity to analyze possible transcriptome modifications. The rat model has made enormous contributions to the understanding of physiological functions and is now the predominant experimental model in cardiovascular research (reviewed in Ref. 23). Furthermore, an increasing amount of sequence data is now available for this species (21, 42). A total of 170,000 rat expressed sequence tags (ESTs) and 7,000 complete rat cDNAs are available in National Center for Biotechnology Information databases compared with 2,000,000 human ESTs and 48,000 complete human cDNAs. These resources make it possible to use a variety of different techniques, such as microarrays (48, 29), representational difference analysis (RDA) coupled to cDNA microarrays (55), and/or differential display (DD) RT-PCR (28) to study transcriptome changes. The transcriptome profiling was yet limited to a few sequences for transplanted rat heart (52, 10) but indicated the reexpression of embryonic transcripts, as expected from the plasticity of the cardiac phenotype (40, 41). As the embryonic transcriptome involved included more than 50% of novel genes (18), we decided to use a differential technique, which can be suitable both for profiling and for the identification of novel genes. RDA coupled to cDNA microarrays and DD-RT-PCR are both appropriate for such an approach. These are of course limited in the number of sequences to be investigated compared with standard microarrays. We chose to use the DD-RT-PCR approach, as we had already developed a quick method for the validation of differential sequences by radioactive RT-PCR (31, 2, 46).

The differential display of mRNAs can demonstrate gene expression differences between two complex biological systems (review in Ref. 53). DD-RT-PCR can be used to look for mediators involved in the phenotype changes that occur in the heart after transplantation. Russell et al. (43) and Utans et al. (52) have validated this approach. The study by Utans et al. (52) used DD-RT-PCR in a rat model of heterotopic heart transplantation to compare mRNAs from allogeneic heart transplants (showing signs of chronic rejection) and syngeneic heart transplants (histologically normal). The results showed that five genes, three known and two unknown, were specifically upregulated in the allografts, suggesting that they may mediate chronic rejection.

The same rat model of heterotopic heart transplantation was used by Depre et al. (10) to determine whether mechanical underload was associated with fetal gene reactivation in heart transplants. In this study, quantitative RT-PCR was used to compare expression of growth factor genes, protooncogenes, myosin heavy chain genes, and the genes encoding two metabolic proteins in overloaded hypertrophic left ventricles and underloaded transplanted left ventricles. Fetal gene reactivation was demonstrated in both situations. We are not aware of any other studies into the genetic underpinnings of the heart transplant phenotype in the absence of chronic rejection. The study by Depré et al. (10) provided the first evidence that distinct gene families may be involved in transplant phenotype reprogramming.

However, the spectrum of genetic modifications associated with heart transplantation is probably very broad. A preliminary estimate of the size and diversity of this spectrum is required to define a transplant-specific transcription pattern that could be compared with expression motifs identified in various fields of functional genomics (12, 26).

In this report, using a DD-RT-PCR approach, we sought to identify specific mRNAs whose expression differs between heterotopic syngeneic heart transplants and native hearts in rats. We selected the right atrium as the target tissue for our study, because it is the most abundantly innervated heart chamber, a feature likely to increase our chances of evidencing denervation-related changes. To focus our study on stable gene expression changes, we studied only the differences in mRNA expression found both 2 days and 7 days after transplantation. We investigated ~2–3% of the expected cardiac transcriptome and found 37 differentially expressed cDNAs. Based on the results of sequencing and sequence homology analysis, these 37 cDNAs were categorized into 3 groups, as follows: 7 unknown transcripts, 16 ESTs, and 14 known mRNAs. Among the known mRNAs, 13 were confirmed by relative RT-PCR analysis as differentially up- or downregulated in the right atria from transplanted hearts compared with native hearts. The corresponding genes belonged to a wide variety of functional groups, encoding proteins involved in energy metabolism, protein synthesis, signal transduction, and gene transcription. The changes in the expression of these genes demonstrated in the transplanted hearts may derive from two major mechanisms, cardiac denervation and hemodynamic underloading, and our results are discussed as a function of these two major determinants. A further interpretation of our results stems from a comparison of the transplant-specific transcription pattern found in our study with the transcription pattern identified by Lee et al. (27) in senescent mouse skeletal muscle. This comparison demonstrated profile homologies suggesting that the phenotype changes observed in heart transplants share similarities with those seen in the aging muscle and that common molecular mediators may be responsible for the coordinate decrease observed in the expression of similar classes of functional genes in senescent muscle and heterotopic heart transplants.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Animals.
Male Lewis rats were used. The animals received humane care in compliance with the Principles of Laboratory Animal Care issued by the National Society for Medical Research and with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 80-23, revised 1978). The experiments were conducted at an Institut National de la Santé et de la Recherche Médicale (INSERM) laboratory that complies with the requirements of the French Ministry of Agriculture and is authorized to perform experiments on live animals (executive order no. 006027, June 10, 1994).

Donor heart collection and heart transplantation.
The whole surgical procedure was done as described in Ref. 38. Donor rats (mean body weight, 277 ± 5 g) were anesthetized with 0.3 ml/100 g im of a mixture (8.5/1.5, vol/vol) of ketamine (50 mg/ml) and chlorpromazine (5 mg/ml), given heparin (1,000 U iv), and ventilated mechanically through a tracheotomy at a rate of 62 breaths/min and at a pressure of 10 mmHg. A midline abdominal incision was performed, and the infrarenal aorta was catheterized in the retrograde direction with an olive-tipped needle (ID 0.6 mm), which was advanced up to the aortic arch and used for subsequent induction of cardioplegia. The chest was opened, and the two superior venae cavae and arterial brachiocephalic trunk were dissected and ligated. Next, the left atrium was incised, and the pulmonary artery was transected at its bifurcation to avoid cardiac distension during administration of the cardioplegia solution. The aortic arch was tied on the catheter, which was used to infuse 20 ml of cardioplegic preservation solution (Celsior) (32) at a constant pressure of 60 mmHg, for 4–5 min at 4°C. The ascending aorta and main pulmonary artery were transected, the pulmonary veins were ligated en masse, and the heart was isolated. The heart was placed rapidly in a sterile glass container filled with 30 ml of the same preservation solution at 4°C, then stored for 4 h at an average temperature of 6°C.

The recipient rats (mean body weight, 229 ± 4 g) were anesthetized in the same manner as the donors but received 100 U/kg heparin. A midline abdominal incision was performed, and the abdominal aorta and inferior vena cava were exposed. The donor heart was removed from the flask stored at 6°C and gently flushed with 20 ml of cold (4°C) saline through the ascending aorta. Then, the donor aorta and pulmonary artery were anastomosed end-to-side to the abdominal aorta and inferior vena cava of the recipient rat, respectively, using 10-0 polypropylene sutures, as described by Ono and Lindsey (37). Throughout reimplantation, the donor heart was kept moist with a cold saline-soaked gauze. The rats were allowed to recover from the anesthesia in individual incubators at 25°C. Duration of the transplant procedure was ~25 min.

Total RNA preparation.
The native and transplanted hearts were harvested 2 and 7 days after transplantation and transferred to an ice-cold saline solution. After removal of the fat tissue, right laterotracheal ganglia, and right phrenic nerve, the right atria were excised and immediately frozen in liquid nitrogen. RNA was extracted using a procedure modified from Chomczynski and Sacchi (6). Frozen myocardial tissue was homogenized in Trizol reagent (GIBCO-BRL, Life Technologies) using a Polytron (Kinematica, Lucerne, Switzerland). After extraction, RNA aliquots were treated with DNase I (MessageClean Kit from GenHunter) prior to phenol/CHCl3 (3:1) extraction and ethanol precipitation in the presence of sodium acetate (0.3 M). The RNA was suspended in DEPC-treated water, and its concentration was assessed using spectrophotometry. Finally, RNA pools were prepared, aliquoted, and stored at -80°C.

DD-RT-PCR.
DD-RT-PCR was performed as previously reported (31) using right atrial RNAs pooled from 12 syngeneic heart transplants [6 harvested 2 days after transplantation (T2 hearts) and 6 harvested 7 days after transplantation (T7 hearts)] and from the 12 native hearts of the transplant recipients [6 harvested 2 days (N2) and 6 harvested 7 days (N7) after transplantation]. The total RNA (200 ng) was subjected to reverse transcription using Moloney murine leukemia virus (MMLV) reverse transcriptase and one of three single-base anchored oligo-dT primers [H-T11A, H-T11G, and H-T11C, RNA Image kit from GenHunter (28)]. One-tenth of the reverse transcription products was subjected to PCR amplification using {alpha}-35S-labeled dATP (1,000 Ci/mmol, Amersham) with 5' primers that were arbitrary 13-mers (H-AP1 or H-AP3, RNA Image kit set 1, GenHunter) and 3' primers that matched those used in the cDNA synthesis. RT and PCR reactions were carried out using a Perkin-Elmer model 9600 thermal cycler, with the reaction conditions and PCR cycling parameters recommended by the manufacturer. The radioactive DNA amplification products were separated on a denaturing 6% polyacrylamide gel (Sequagel-6, National Diagnostics). RT, PCR, and electrophoresis were repeated twice. Each display reaction (defined by 1 of the 6 primer combinations used) included a parallel analysis of the two time groups (hearts harvested 2 and 7 days after transplantation), in which two samples of each RNA pool (N or T) were analyzed in adjacent lanes. Candidates for differential up- or downregulation were defined as those DNA duplicate bands visually identified as more intense or less intense, respectively, in all atria from the syngeneic transplants, compared with all atria from the native hearts of the transplant recipients, at both time points. Gel bands of interest were excised from sequencing gels, reamplified using the same primer set as in the display reactions, and subcloned into PCR-TRAP cloning vectors using the PCR TRAP Cloning System (GenHunter). cDNA inserts ranging from 50 to 600 bp were sequenced by Genome Express (Paris, France) using the Lseq or Rseq primers. The nucleotide sequences obtained were compared with known sequences by matching to entries in the GenBank, EMBL, and EST DNA databases.

Relative RT-PCR analysis.
We used aliquots of N7 and T7 RNA pools to confirm the differential display patterns by performing specific radioactive RT-PCR amplifications of presumptive differential or 18S rRNA sequences, using a protocol modified from Elalouf et al. (13) and described previously (20). The specific primers used for the 18S rRNA reactions, as well as those used for the presumptive differential sequences, were designed using Oligo 4S software and are shown in Tables 1 and 2; the primers used for the presumptive differential sequences were either nested within or were overlapping the ends of the differential display fragments, with the exceptions of T8 bis and T19 bis. The 3' primer, which was included in the RT reaction, was selected for minimal self-priming, and both the 3' and the 5' primers were 20- to 30-mers in length. Pilot reactions were used to check the specificity of the amplification products and the range of total RNA concentrations used in the relative RT-PCR reactions. RT-PCR products were analyzed on a 6% polyacrylamide sequencing gel (Sequagel-6, National Diagnostics). The gel was dried and exposed to a phosphorimager screen for quantification of the radioactive signals using a Fujix Bio-imaging Analyzer System (BAS 1000). RT-PCR amplification using specific 3' and 5' 18S rRNA primers (Table 1) was performed as a control to assess differences in total RNA concentrations between N7 and T7 RNA pools. Normalized values of presumptive differential expressions were obtained by dividing the corresponding radioactive signals by the mean 18S rRNA signals obtained from triplicate comparisons of individual RNA samples, and differences between transplanted vs. native tissues were assessed using one-way ANOVA. The threshold for statistical significance was set at 5%. For each transcript, expression in the transplants was expressed as the percentage of the mean value in the native hearts.


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Table 1. RT-PCR amplification of standard sequences

 

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Table 2. RT-PCR amplification of differential candidates

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Identification of differentially expressed cDNAs.
Transplanted and native right atria harvested on posttransplantation days 2 (T2 and N2) and day 7 (T7 and N7) were included in each of our differential display experiments. Figure 1 shows our experimental strategy, and Fig. 2 shows a representative display pattern. Comparison of transplanted and native atria identified 44 transplant-specific cDNA bands present at both time points, nine present only on day 2, and five present only on day 7 (Fig. 3). Only the 44 transplant-specific bands present at both time points were subjected to further studies; 20 of these bands indicated gene upregulation, and 24 indicated gene downregulation.



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Fig. 1. The experimental approach used to identify transcripts differentially expressed in right atria (RA) from heterotopic syngeneic heart transplants (T) and native hearts (N) in rats, and to define a transplant-specific differential mRNA pattern by validating the differential expression of known specific mRNAs or genomic sequences. LV and RV, left and right ventricles, respectively; EST, expressed sequence tag.

 


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Fig. 2. Differential display comparing right atrial RNA pools from heterotopic syngeneic heart transplants and native hearts in the same rats. Right atrial total RNA pools (6 atria in each group) from native and transplanted hearts 2 days after surgery (N2 and T2, respectively) and 7 days after surgery (N7 and T7, respectively) were subjected to differential mRNA analysis. Duplicate cDNA samples from each cardiac RNA pool were analyzed in parallel with an RNA-free control (lane 0) and with brain cDNA (lane B). Autoradiographs of PCR products (after electrophoresis in 6% polyacrylamide gels) are shown for three different primer combinations. Arrows, bands differentially displayed in the right atria from T2 and T7 hearts identifying cDNA candidates for downregulation (bands 1, 2, and 4) or upregulation (bands 3, 5, and 6) in the transplants.

 


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Fig. 3. Distribution of gel bands differentially expressed in the right atria of heterotopic syngeneic transplants as a function of time since transplantation or nucleotide sequence information. Nine bands were differentially displayed 2 days posttransplantation only, and five bands were differentially displayed 7 days posttransplantation only. Thirty-seven bands were differentially displayed at both time points. These 37 bands corresponded to distinct cDNA sequences, which were subjected to sequencing and sequence homology analysis. Based on the results, these 37 sequences were categorized as ESTs (n = 16), completely or partially characterized mRNAs or genomic sequences (n = 14, of which 13 were validated), and unknown sequences (n = 7).

 
The differentially expressed cDNAs were reamplified and subcloned prior to sequencing and to homology sequence identification using the BLAST algorithm. Based on sequence identity or homology with known expressed sequences available in public databases, the 44 differentially expressed cDNAs present at both time points were categorized into three groups, as follows: 18 cDNAs were identical or highly homologous to known transcripts (known mRNAs or genomic sequences), 19 were identical or homologous to ESTs, and 7 showed no significant homology with database sequences. Of the 18 cDNAs matching known transcripts, 11 were found once and three were found two or three times. After elimination of redundant sequences, the number of known transcripts was 14. Similarly, three redundant cDNAs were found in the EST group, leaving 16 distinct EST sequences. Thus the total number of differentially expressed cDNAs found at both time points was 37 (Fig. 3).

The unknown gene and EST expression pattern.
Seven cDNAs (5 up and 2 down differential candidates) ranging between 121 and 271 bp corresponded to unknown genes. Among the 16 ESTs, 9 matched with up- and 7 with down-displayed candidate cDNAs that ranged between 87 to 579 bp. Three from the 16 EST sequences have been cloned from mouse or rat embryonic tissues, 1 was cloned from PC12 cells, and the remaining 12 have been cloned from adult mouse or rat tissues. The nucleotide identity between candidate differential cDNAs and the corresponding ESTs vary from 64% to 100%, with 14 nucleotide sequences displaying 95–100% identity.

Relative RT-PCR to confirm differentially expressed transcripts.
Only the 14 transcripts matching known transcripts were subjected to this study. These transcripts were selected as present on both day 2 and day 7 after transplantation. Total RNA pools from atria harvested on day 7 were used for the confirmation studies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was evaluated as a possible constitutive control in relative RT-PCR experiments, using the specific primers given in Table 1. The levels of GAPDH mRNA appeared to be downregulated in total RNA from transplanted atria (Fig. 4). This finding was confirmed after normalization of the GAPDH signal for an 18S ribosomal signal obtained after RT-PCR amplification of the same RNA pools. The normalized expression of GAPDH mRNA was decreased by more than 50% in the transplants compared with the native hearts. In contrast, when several identical samples and different RNA concentrations were tested in parallel, RT-PCR amplification of 18S ribosomal RNA (using the primers in Table 1) did not show significant differences between native and transplanted RNA pools (Fig. 4). Therefore, we used 18S ribosomal RNA as the constitutive control for all the differentially expressed transcripts analyzed by relative RT-PCR in the present study.



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Fig. 4. RT-PCR amplification of 18S rRNA and GAPDH mRNA from the right atria of heterotopic syngeneic heart transplants and native hearts in the same rats. Triplicate preparations of total RNA pools from native and transplanted hearts collected 7 days after surgery were amplified using the specific primer sets listed in Table 1. After polyacrylamide gel electrophoresis of PCR products, the specific radioactive signals were converted into arbitrary densitometric values by two-dimensional image analysis and compared between the native and transplanted groups. M, molecular weight marker. B, RT-PCR reaction blank.

 
Relative RT-PCR analysis of differentially expressed mRNAs matching known transcripts was performed using an experimental pattern that was similar for each known transcript to the pattern used for ribosomal RNA, except that the total RNA concentration varied as a function of the relative abundance of the transcript being tested, from 2.5 to 30 ng, according to the results of pilot experiments. The nucleotide sequence of each primer pair and the annealing temperature of each of these specific reactions are given in Table 2. The results of this analysis (Table 3) confirmed significant differences between the transplant and native samples in the level of expression of 13 of the 14 transcripts, when primers nested within or overlapping the ends of the amplicon initially identified in DD-RT-PCR experiments were used (Table 2). The radioactive signals identifying the PCR products from these 13 transcripts are shown in Fig. 5. The remaining cDNA, identified based on nucleotide identity (197/199) as encoding the rat band 3 Cl-/HCO3- exchanger mRNA [cloned by Kudrycki et al. (25)], was initially selected as an updisplayed gel band but was shown by RT-PCR analysis to be downregulated in transplant samples (T/N ratio, 0.32; P < 0.05).


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Table 3. Specific transcripts validated as downregulated and upregulated in the right atria from grafted hearts

 


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Fig. 5. Relative RT-PCR analysis of partially or completely characterized mRNAs identified by DD-RT-PCR in samples of right atria from heterotopic syngeneic heart transplants. Total atrial RNA pools from native and transplanted hearts harvested 7 days after surgery were used at the concentrations indicated (in ng per assay) for specific RT-PCR reactions. For each differential band [T6 to G8(25), A; T19 to G35(42), B], the set of specific primers listed in Table 2 was used. Specific primers were selected within or overlapping the ends of the differential display cDNA fragments; exceptions to this were T19(bis) and T8(bis) (B), for which primer sets selected within the coding parts of the corresponding transcripts were used. Specific transcripts are shown that were validated as downregulated (A) or upregulated (B) in the transplants based on statistical comparisons between normalized mean specific signals in the transplants (T) and native hearts (N). For each transcript, the mean significant difference is given as the T/N ratio at the left and in Table 3. M, molecular weight marker. B, RT-PCR reaction blank.

 
For two of the confirmed differential transcripts, no differential expression was detected using primers selected within the coding part of the transcript. This confirms that the transcript region selected as the target has a considerable impact on the results of relative RT-PCR analysis. Thus T8 mRNA was 47% upregulated in the transplants when primers (T8 primers, Table 2) from the 3'-untranslated region (3'-UTR) of the transcript (within the original DD-RT-PCR 250-bp amplicon) were used; conversely, no differential expression was found using T8 bis (Table 2) primers, which amplify part of the coding region [nucleotides 387–990 in the sequence cloned by Fujioka et al. (17)]. Similarly, 27% upregulation of T19 mRNA was demonstrated in the transplants when T19 primers (Table 2) located within the 3'-UTR initially amplified in DD-RT-PCR reactions were used, whereas no differential expression was evident with T19 bis (Table 2) primers selected within the coding region of the transcript [nucleotides 1602–2280 of the rat sequence cloned by Schwarzbauer et al. (49)].

Two distinct cDNAs, G'34(39+40), encoding the same transcript but homologous to different regions of this transcript, were identified in the subclones of one of the initial downdisplayed gel bands. These two cDNAs were probably produced by cleavage of a cDNA strand during reamplification and subcloning.

As shown in Table 3, the magnitude of the change in expression of the 13 mRNAs differentially expressed in the transplanted atria compared with the native atria varied from about -50% (T6 downregulation) to +190% (G35 upregulation). However, for 10 of the 13 mRNAs, the difference was 10–50% in either direction.

For the 13 differentially expressed mRNAs, Table 3 shows the position of the DD-RT-PCR amplicon within the matching mRNA or genomic sequence found in a database. For 9 of these 13 mRNAs, the DD-RT-PCR amplicons were located within the 3'-UTR, consistent with RT reaction initiation by anchorage within the poly(A) tail.

We compared the relative levels of 11 transcripts in RNA pools from native and transplanted atria on days 2 and 7 (Table 4). For these 11 mRNAs, the direction of the differential change was identical in transplanted atria from day 2 and day 7 pools, but their amplitude of change was smaller at day 7 (Table 4).


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Table 4. Differential expression of 11 specific transcripts in transplanted right atria harvested 2 days after surgery

 
We investigated whether pooled RNAs could be contaminated by an abnormal (ischemic or thrombosed) specimen, by comparing the relative levels of two distinct different transcripts in individual samples. We selected a downregulated transcript, that for the mitochondrial proton/phosphate symporter [G26(6); Table 3], and an upregulated transcript, that for adenine phosphoribosyltransferase [G13(6); Table 3]. The expression of these specific transcripts was differentially regulated in the individual specimens removed from animals undergoing heart transplantation on day 7 (Table 5). The directions and amplitudes of change were similar to those observed with the pooled day 7 samples.


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Table 5. Differential expression of two specific transcripts in individual right atria harvested 7 days after surgery

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using 6 primer combinations, we explored about 600 transcription bands displayed on DD-RT-PCR gels. Among these bands, 44 were differentially expressed in transplants and native hearts. After elimination of redundancies, we were left with 37 cDNAs, among which 33 (75%) matched GenBank entries, 19 (43%) uncharacterized ESTs, and 14 (32%) known genomic sequences. These 14 transcripts were subjected to confirmation of differential expression by relative RT-PCR analysis. The use of DD-RT-PCR is obviously not optimal to give a complete profile of transcriptional changes. Compared with microarrays that can cover 6,000 to 9,000 cDNAs, we investigated 600 transcription bands corresponding to transcribed sequences, accounting for a small but a significant fraction, of at most 10% of the transcriptome covered by microarrays. The limitations of DD-RT-PCR include: 1) difficulties of quantifying the magnitude of gene expression, 2) dependence on primer efficiency and specificity; and 3) need for bona fide reference markers. In the present study, we used radioactive RT-PCR based on a specific primer used in the reverse transcription step to validate differential expression candidates (2, 31, 46). This technique confirmed that 13 of the 14 transcripts resulted from differential expression. Levels of standard reference markers, such as GAPDH, have been shown to vary during embryonic development, whereas 18S rRNA levels do not, and we therefore chose 18S rRNA as a reference marker (2).

The experimental model of rat cardiac heterotransplantation suffers from some important limitations because it is underloaded and denervated. However, this syngenic graft in the same hormonal environment as the native heart can be studied for several days to address physiopathological questions that cannot be tackled in vitro. Unlike Depre et al. (10), who carried out a heterotopic heart transplantation procedure in 60 min, we used a 25-min operative procedure preceded by a 4-h cold incubation at 6°C, to mimic the human heart transplantation process (38). This paradigm may give rise to potential confounding components. However, we studied only transcripts that were differentially regulated both 2 days and 7 days after transplantation, which may account for the low false-positive rate in our study. Furthermore, we compared transcript level variations at day 2 and day 7 for 11 differentially expressed genes (Table 4). This comparison did not evidence the large changes one can expect if transient ischemic episodes have followed the transplantation. The variations of transcript ratio between day 2 and day 7 may reflect individual variability, as documented in data from individual right atria heart (Table 5).


Heterotransplantation induces subtle changes in gene expression.
The transplant-specific repression or induction ratios (Table 3) ranged from values of 0.51 for developmentally regulated cardiac factor-5 (DRCF-5) mRNA repression to 2.9 for glutaredoxin mRNA induction (Table 3). For many of the differentially expressed transcripts, the change in expression compared with the native hearts was small, suggesting that subtle changes in gene expression may be an important aspect of cardiac adaptation to transplantation. Furthermore, changes that occur in vivo are more likely to be subtle than those observed in isolated cells transiently stimulated by drugs. For instance, large changes in gene expression have been evidenced between unstimulated and serum-stimulated fibroblasts (22) and upon activation of a T cell clone by an anti-CD3 antibody (1).

Cardiac gene expression changes in hemodynamic unload are not restricted to the re-expression of a fetal gene program.
Using heterotopic rat heart transplants, Depre et al. (10) evidenced phenotype changes consisting in the re-expression of a fetal gene program. In our experiments, we found that fibronectin was differentially expressed, in agreement with reexpression of a fetal form, as already described during pressure overload (47, 45). Fibronectin was identified using relative RT-PCR analysis with primers located within part of the 3'-UTR. No differences in regulation were demonstrated with primers located within the coding region of this transcript. These findings were suggestive of differential expression of splicing variants as the mechanism underlying the upregulation of fibronectin mRNA. Thus the fibronectin gene activation demonstrated in the transplanted atria in our study strongly suggests that fetal variants of this gene were expressed in the heterotransplant and that hemodynamic underload could account for the differential expression of this gene. However, the majority of the differentially regulated transcripts identified in the present study are not known to be developmentally regulated.

Denervation is a potential determinant of differential gene expression.
In vertebrate skeletal muscle, innervation plays a role in maintaining normal fiber numbers, and nerve-dependent shifts in fiber type are commonly observed during maturation, illustrating the ability of the nerve supply to modify the muscle fiber phenotype (review in Ref. 19). Numerous biochemical changes occur in mammalian skeletal muscle following denervation (11, 34). These changes include metabolic alterations that reflect depressed energy production, such as decreased levels of several enzymes involved in oxidative metabolism, decreased oxygen consumption, decreased metabolic rate, and altered mitochondrial integrity (references in Ref. 11), suggesting that the downregulation of the four genes associated with energy metabolism (glycogenin, GTP-specific ß-subunit of SCS, ANT, and proton/phosphate symporter), that were downregulated in the transplanted right atria, may be secondary to cardiac denervation.

We found evidence of stannin (Sn) mRNA downregulation in our transplanted right atria. Its neuronal expression described by Toggas et al. (50) and Dejneka et al. (9) suggests that its downregulation within transplanted atria may reflect cardiac denervation.

Transplantation and aging share transcriptional regulation homologies.
A further interpretation stems from a comparison of our heart transplant mRNA profile with the gene expression profile reported by Lee et al. (27) as characteristic of senescent mouse skeletal muscle. This comparison is based on results obtained with experimental approaches differing considerably in both their design (arbitrarily displayed subsets of transcripts in the DD-RT-PCR method, preselection of oligonucleotide sets as an antecedent before array fabrication) and the fraction of the transcriptome investigated [~600 differential bands in our analysis vs. 6,347 genes studied by Lee et al. (27) with microarrays].

Lee et al. (27) demonstrated age-related alterations in the expression of 113 genes (1.8% of the gene representation). Aging was associated with an increase in the expression of genes involved in the stress response and in neuronal growth and with a coordinate decrease in the expression of genes involved in energy metabolism (including those associated with the mitochondrial function) and in biosynthesis (including protein turnover) (27).

One similarity between the senescent and transplant profiles is the higher proportion frequency of transcriptional downregulation found, as in our study, for the 45 specific genes that displayed the largest alterations changes in the aging profile reported by Lee et al. (27). More important elements of similarity between the two profiles concern the coordinate downregulation of two classes of genes, those involved in energy metabolism and in protein synthesis.

The energetic expression motif.
In their expression pattern, Lee et al. (27) reported downregulation for four transcripts (those for {alpha}-enolase, phosphoprotein phosphatase, IPP-2, and glucose-6-phosphate isomerase) encoding proteins involved in a functional metabolic class denoted as glycolysis and glycogen metabolism. These age-related decreases in the expression of genes involved in energy metabolism also included changes in the transcription of genes associated with mitochondrial function and turnover; two transcripts of this subgroup, involved in mitochondrial bioenergetics, ATP synthase A chain, and NADP transhydrogenase, were downregulated in the senescent tissue.

In our subgroup of transplant-specific downregulated mRNAs, two transcripts were products of genes involved in energy metabolism: the glycogenin mRNA [the encoded protein primes the synthesis of glycogen by its self-glycosylating activity (30)] and the SCS mRNA [encoding a GTP-specific ß-subunit of succinyl-CoA synthetase, which catalyzes the substrate-level phosphorylation step of the citric acid cycle (44)]. In addition, two downregulated transcripts were found to encode mitochondrial proteins involved in energy metabolism: the ANT mRNA [encoding the mitochondrial adenine nucleotide translocator, which determines the rate of ADP/ATP flux between the mitochondrion and the cytosol, thereby regulating the dependency of cells on oxidative energy metabolism (36)], and the mitochondrial proton/phosphate symporter mRNA [encoding a protein responsible for the energy-coupled translocation of inorganic phosphate across the inner mitochondrial membrane, an essential step that is crucially involved in oxidative phosphorylation (15)].

The protein biosynthesis motif.
According to Lee et al. (27), aging led to large decreases in the expression of genes associated with biosynthetic activities. This reduction was accompanied by a concerted decrease in the expression of genes encoding proteins involved in protein synthesis or turnover, and six transcripts (EF-1{gamma}, 20S proteasome subunit, ubiquitin thiolesterase, 26S proteasome component TBP1, and Unp ubiquitin-specific protease) that belonged to this functional class were identified in the senescent profile (27). We found that two mRNAs, encoding the ribosomal proteins L27A (54) and S6 (5), which are directly involved in the regulation of protein turnover, were downregulated in the cardiac grafts.

Therefore, the comparison of transplant and senescent profiles clearly showed that two functional classes of genes were subjected to a similar concerted downregulation in these two profiles, demonstrating expression motif homologies in transcription patterns between the senescent muscle and the transplanted cardiac atrial chamber.

As our experimental paradigm induces a hemodynamically unloaded heart, it would be important to investigate a possible correlation between the senescent-like expression pattern and this unloading parameter.

Conclusion.
In the present study, we investigated the consequences of cardiac heterotransplantation upon the transcripts expressed in the right atrial tissue. We found that heterotransplantation induces transcript changes in a variety of gene families, at the difference of the report by Depre et al. (10). Furthermore, the diversity of genetic modifications that are associated with cardiac transplantation in this cardiac chamber defined a transplant-specific transcription pattern with unexpected homologies with senescent skeletal muscle phenotype.


    ACKNOWLEDGMENTS
 
The Celsior preservation solution was a generous gift from Pasteur Mérieux Sérums et Vaccins (Marcy L’Etoile, France).

This work was supported by grants from the INSERM. We are very grateful to Dr. Abdeslam Oubénaïssa for performing the heart transplant procedures and for providing the expert surgical assistance that made this work possible.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: J.-M. Moalic, U127 INSERM, Hôpital Lariboisière, 41 Bd de la Chapelle, 75475 Paris cedex 10, France (E-mail: jean-marie.moalic{at}inserm.lrb.ap-hop-paris.fr)


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