Identification of Candidate Genes Potentially Relevant to Chamber-Specific Remodeling in Postnatal Ventricular Myocardium

Molecular predisposition of postnatal ventricular myocardium to chamber-dependent (concentric or eccentric) remodeling remains largely elusive. To this end, we compared gene expression in the left (LV) versus right ventricle (RV) in newborn piglets, using a differential display reverse transcription-PCR (DDRT-PCR) technique. Out of more than 5600 DDRT-PCR bands, a total of 153 bands were identified as being differentially displayed. Of these, 96 bands were enriched in the LV, whereas the remaining 57 bands were predominant in the RV. The transcripts, displaying over twofold LV-RV expression differences, were sequenced and identified by BLAST comparison to known mRNA sequences. Among the genes, whose expression was not previously recognized as being chamber-dependent, we identified a small cohort of key regulators of muscle cell growth/proliferation (MAP3K7IP2, MSTN, PHB2, APOBEC3F) and gene expression (PTPLAD1, JMJD1C, CEP290), which may be relevant to the chamber-dependent predisposition of ventricular myocardium to respond differentially to pressure (LV) and volume (RV) overloads after birth. In addition, our data demonstrate chamber-dependent alterations in expression of as yet uncharacterized novel genes, which may also be suitable candidates for association studies in animal models of LV/RV hypertrophy.


Introduction
Ventricular (or cardiac) remodeling is commonly defined as a physiological or pathological process that can occur under various conditions of pressure/volume overload. A common feature of ventricular remodeling is hypertrophy of the cardiomyocytes. The type of cardiac workload determines the pattern of ventricular hypertrophy: volume overload induces eccentric, while pressure overload induces concentric remodeling. Under various pathological conditions, compensatory concentric hypertrophy can lead to eccentric hypertrophy, dilatory ventricular remodelling, and heart failure (reviewed in [1,2]). The molecular signature of concentric versus eccentric hypertrophy, although poorly defined as yet, is nevertheless of critical relevance in cardiac basic and clinical research [3][4][5][6][7][8].
The early neonatal heart is a conventional model for the study of distinct patterns of ventricular hypertrophy (i.e., concentric versus eccentric). At birth, cardiomyocytes begin to enlarge in response to the demands of physiological workload, as opposed to processes driven predominantly by developmental mechanisms. Particularly, the left ventricle (LV) is exposed to a higher-pressure overload in comparison to the right ventricle (RV), which is exposed to a relatively higher-volume overload. As a result, the LV undergoes rapid concentric hypertrophy, while the RV undergoes eccentric hypertrophy associated with dilatory RV-chamber remodeling. Our previous data revealed differences in the expression of cardiac ankyrin repeat domain 1 factor (ANKRD1/CARP) between the LV and RV before the appearance of morphologically identifiable signs of LV-concentric or RV-eccentric hypertrophy in newborn piglets [9]. Other research reported certain LV/RV-specific metabolic differences in normal and ischemic newborn piglet heart [7]. We interpreted these results as reflecting a certain type of molecular predisposition of newborn ventricular myocardium to LV-concentric and RV-eccentric remodeling during postnatal development.  In the present study, we focused on large-scale transcriptomic analysis to compare differences in gene expression levels in the LV versus RV in newborn piglets. Given that commercially available DNA microarray platforms suitable for performing transcriptional profiling in pig are still poorly developed, we conducted comparative LV versus RV gene expression profiling in newborn piglets using mRNA differential display (DDRT-PCR). In addition, unlike microarraybased platforms, DDRT-PCR can be used to detect expression changes in both known and novel transcripts including alternate splice variants [10]. This approach allowed us (1) to perform an unbiased assessment of genes which expression is predominantly associated with piglet LV or RV myocardium and (2) to distil a large body of expression data into a discrete set of candidate genes for which regulation was not previously recognized as being chamber-dependent. Further studies on these differentially regulated genes will likely lead to the identification of additional novel gene families and pathways involved in the chamber-dependent response of ventricular myocardium to a variety of physiological and pathological stimuli.

Animals and Tissue
Sampling. Animals were treated and cared for in accordance with the European commission directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes, and all animal protocols were approved by the ethical research committee of Galicia (Spain). Newborn (10-12 hours after birth) Large White piglets were obtained from a local commercial breeder (La Coruña, Galicia) and maintained in an automatic nursery system (Nütinger System). The newborn and 20day-old animals were anaesthetized, the thoracic cavity was opened through a median sternotomy, and the entire heart was rapidly removed, weighed, and photographed while still beating. Then the isolated heart was placed on an ice-cold petri dish, partially sectioned at the midpoint of the LV length and photographs of the open ventricular chambers were taken (Figure 1). Immediately after this step, the LV and RV free walls were dissected, flash frozen in liquid nitrogen, and stored at −80 • C until study.

RNA Isolation.
Deep-frozen tissue samples (100-150 mg), encompassing the full thickness of the free wall of the LV and RV ventricle, were directly disrupted in RLT buffer (Qiagen) using a high-speed rotor-stator homogenizer (Ultra-Turrax T8, Germany), digested with Proteinase K (Qiagen), loaded onto a RNeasy Midi column (Qiagen), subjected to on-column digestion of DNA with RNase-free DNase (Qiagen) and the analysis proceeded in accordance with the manufacturer's recommendations. Resulting RNA preparations were ethanol-precipitated, resolved in RNasefree water, and kept at −80 • C. RNA yield and purity was determined spectrophotometrically at 260-280 nm and RNA integrity was verified by running samples on 1.5% agarose gels and staining with ethidium bromide.
2.3. Differential Display mRNA Analysis. The reverse transcription-PCR differential display (DDRT-PCR) analysis was performed as described [11] with minor modifications [12]. To yield starting material for the DDRT-PCR, total RNA preparations independently isolated from the LV and RV of three newborn piglets were, respectively, pooled at equal ratios, and 4 μg of RNA was reverse transcribed using the SuperScript III (Invitrogen) and T7-oligo-dT primer. Pooled first-strand cDNAs were amplified side-by-side by PCR using 230 different primer combinations (10 two-baseanchored oligo-dT and 23 arbitrary primers purified by HPLC, Table 1).
Nontemplate (NT) and non-RT RNA (N-RT) template reactions were used as negative controls. In each DDRT-PCR set-up, reactions were performed at least in duplicate to test whether differences in LV/RV gene expression are likely to be real. PCR was performed, using the AmpliTaq DNA polymerase (Invitrogen), under the following conditions: initial denaturation (94 • C, 2 minutes), stage I (5 cycles, each of which included: 94 • C, 30 seconds; 40 • C, 1 minute; 72 • C, 1 minute), stage II (25 cycles, each on which included: 94 • C, 30 seconds; 50 • C, 1 minute; 72 • C, 1 minute), and final extension (72 • C, 10 minutes), sample store at 6 • C. PCR-amplified products were subjected to fractionation on 8% polyacrylamide gels (PAAG) (Mini-Protean-III, Bio-Rad) and fluorescently stained by SYBR Green I (Sigma). Image acquisition and intensity of bands were estimated by densitometry (VersaDoc 1000) and Quantity One software (Bio-Rad). Differentially regulated amplification products were defined as those bands that were similarly displayed at least in two experimental replicates. Using a sharp, sterile razor blade, a rectangular piece of gel corresponding to an individual band of interest on the PAAG was excised and electroeluted (D-tube Electroelution Kit, Novagen). After a short centrifugation, the eluate was transferred to a clean tube. The extracted DNA was used directly as the template for PCR with T7 and M13 reamplification primers (see Table 1). Cycling conditions were as described for DDRT-PCR except stage I at 45 • C and stage II (20 cycles) at 55 • C. After reamplification, each PCR reaction was electrophoresed through a 1.5% agarose gel with ethidium bromide to assure that the correct sized fragment was amplified. Reamplified cDNA fragments were eluted (QIAquick Gel Extraction Kit, Qiagen), cloned into pCRII-TOPO vector (Invitrogen), and sequenced by (Secugen), (Madrid, Spain). The nucleotide sequences obtained were compared with known sequences by searching the GenBank database with BLAST algorithms.

Quantitative RT-PCR.
Differential gene expression was further confirmed by real-time quantitative PCR (qRT-PCR) as described [13] using Bio-Rad IQ5 instrument and Bio-Rad ACAATTTCACACAGGAGCTGAACCGG primers for reamplification of DD bands T7 GTAATACGACTCACTATAGGGC M13rev-48p AGCGGATAACAATTTCACACAGGA * Each anchor primer has T7 sequence (bold) on the 5 end. * * Each arbitrary primer has M13 sequence (bold) on the 5 end.
SYBR Green Mix [14,15]. Whenever possible, the primer pairs were designed to be located in different exons of a given sequence. Individual heart-matched LV/RV cDNAs isolated from three newborn and three 20-day-old piglets were used as templates. Each primer pair used yielded a single peak of dissociation on the melting curve and a single band with expected size on PAAG [12]. A negative NT and N-RT controls were included in each reaction set. Detection of ribosomal protein L19 (RPL19) mRNA was used to normalize the expression of target mRNAs. The efficiency of target and reference amplification was tested and found to be approximately equal. Results were defined as the target genes expression normalized against rpl19 gene expression in both ventricles. Fold changes were calculated using the C T method. Primer sequences and additional details on qRT-PCR are available upon request.

Data
Analysis. Values were expressed as means ± SEM. mRNA expression was quantified using the comparative threshold cycle method. Statistical analyses were performed with the SPSS 13 software. A P value < .05 was considered to be statistically significant.

DDRT-PCR Analysis Allows Reliable Transcriptomic Profiling of Ventricular Myocardium in Newborn Piglets.
For mammalian cells, it was calculated that 20 arbitrary in conjunction with 12 anchored primers would statistically amplify all mRNA sequences [16]. We used 23 arbitrary and 10 two-base-oligo(dT) anchored primers (Table 1), resulting in 230 display primer combinations. A total of about 5,600 distinct cDNA fragments corresponding to genes expressed in piglet LV/RV myocardium were detected. A representative example of DDRT-PCR banding patterns is illustrated in Figure 2(a).
The average number of bands generated by one primer pair was 26, the minimum was 0, and the maximum was 44. About 70% of the primer pairs produced 20-40 bands (Figure 2(b)). Size distribution analysis of cDNA bands generated by DDRT-PCR revealed a minor fraction of shortsized (100-300 nt) bands, while the fragments with a size from 300 to 1,000 nt, which is a preferable choice for cloning and sequencing, made up about 60% of all detected bands (Figure 2(c)).
Taken together, the results indicated that under our experimental conditions, transcript-banding patterns generated by DDRT-PCR could be sufficient for comparative expression analysis of the LV versus RV myocardium of newborn piglets.  Table 2.

DDRT-PCR Profiling Identifies Differentially Expressed Genes in the LV versus RV Myocardium of Newborn Piglets.
Direct side-by-side comparison of the mRNAs between the LV and RV of the newborn piglet heart revealed that the majority of profiled genes (97%) were similarly expressed in both ventricles. Out of more than 5,600 DDRT-PCR bands amplified by the primer combinations used, a total of 153 bands, ranging in size from 300 to 1,000 nt, were identified as being qualitatively differentially displayed. Of these, 96 transcripts were enriched in the LV, whereas the remaining 57 were predominant in the RV. Figure 3 illustrates the relative differential expression of a representative set of bands in the LV as compared to the RV myocardium. Once differentially displayed PCR products were detected, the fragments which displayed over twofold LV-RV expression differences (40 bands) were recovered from gels, reamplified, cloned, and sequenced. The differential expression of these genes was further confirmed using qRT-PCR analysis. In this manner, over 80% (32 bands) of the selected bands were confirmed to be differentially expressed in the two ventricular chambers of newborn piglets ( Table 2). The BLAST searches for sequence similarity revealed that 6 of the 32 cloned cDNA fragments with confirmed differential expression are potentially novel transcripts with no significant match in the current databases, suggesting that they may either encode as yet uncharacterized proteins or correspond to unknown regions of identified genes (untranslated, nonconserved regions). The remaining 26 cDNA sequences were identified by BLAST sequence comparisons as genes related to modulation of gene expression (PTPLAD1, PHB2, CEP290, JMJD1C), regulation of cell growth and differentiation (MSTN, MAP3K71P2, APOBEC3F, PHB2), biomechanical stress sensing and myofibrilar assembly (TTN, ANKRD1), muscle contraction (TNNT2, ACTC1), extracellular matrix remodeling (ADAMTS3, COL1A2), calcium control (SLC8A1), and energy metabolism (ATP5C1, ND6). Table 2 provides details of the extent of relative LV/RV upregulation (fold change) as well as the known function(s) of identified genes. Among the differentially expressed genes, only a small portion displayed over 4-fold expression differences between LV and RV (PTPLAD1, TPM2, ACTC1, ANKRD1, ANKRD1-I8, PDE3A, D162, TNMD, D170). In this sense, chamber-dependent regulation of expression of these known and novel transcripts may be primarily associated with different patterns of postnatal ventricular remodeling.
MSTN (myostatin) characterized by LV-predominant expression in newborn myocardium also stood out as an interesting candidate, given its roles in cell growth and proliferation. Recently, it has been demonstrated that MSTN is a potent repressor of cardiac muscle cell proliferation and growth, and that in vivo loss of MSTN induces eccentric hypertrophy associated with enhanced responsiveness of ventricular myocytes to beta-adrenergic stimulation [17,18]. We, therefore, examined this gene expression in both ventricles at advanced stages of postnatal development when morphological differences between concentric (LV) and eccentric (RV) remodeling become evident, that is in 20day-old piglets (see Figure 1(d)). The LV/RV MSTN mRNA ratio found in newborn piglets (i.e., 2 : 1) was significantly amplified in 20-day-old animals (i.e., 6 : 1) due to MSTN upregulation in the LV of the latter age group, while the gene's expression levels in the RV were similar in two groups studied (Figure 4). The results indicate that in neonatal piglets a process of RV-eccentric remodeling is associated with the same relative low MSTN level as was found in the RV at birth.
Collectively, the comparison of gene expression between the LV and RV shortly after birth, when LV/RV loading conditions are dramatically changed as compared to the latefetal period, demonstrates that such analysis provides clues for identifying hallmark genes whose expression is regulated in a chamber-dependent manner at the earliest stages of postnatal LV-concentric and RV-eccentric remodeling.

Discussion
The DDRT-PCR technique, which was first developed in 1992 [19], is still the method of choice for an unbiased comparison of mRNA expression patterns between samples that are very similar and often results in identification of nonabundant, rare, or novel transcripts [10,20,21].
Using a nonradioactive DDRT-PCR technique, we identified the transcripts that reproducibly showed different expression levels between LV and RV in newborn piglets. These differences do not correlate with either cardiomyocyte cell volume [22] or ventricular wall thickness ( [9]; see also Figure 1(b), this work), which are practically equal in both piglet ventricles during or shortly after birth. Thus, in  Journal of Biomedicine and Biotechnology this system a molecular prepattern precedes the appearance of morphologically identifiable signs of LV-concentric and RV-eccentric hypertrophy. We suggest that the observed differences in gene expression are intrinsic to the distinct molecular makeup of the LV versus RV rather than to their hyperplastic/hypertrophic growth status, which is similar in both ventricles at birth. Further, the content of certain wellknown markers of cardiomyocyte hypertrophy (beta-myosin heavy chain and myosin light chain 2 ventricular) was found to be similar in both the LV and RV of newborn piglets [9]. Moreover, expression levels of the transcriptional cofactor, myocardin, which induces cardiomyocyte hypertrophy [23,24], are equal in both ventricles of these animals [25]. Therefore, it seems reasonable to interpret the differences in gene expression detected in our present work as indicative of an L-R molecular predisposition of the newborn myocardium to respond to dramatic changes of the hemodynamic loads shortly after birth when the LV is exposed to a higherpressure load (concentric hypertrophy promoting condition) in comparison to the RV, which is exposed to a highervolume load (eccentric hypertrophy promoting condition). The vast majority of the transcripts differentially expressed in the LV and RV of newborn piglets correspond to genes which were not previously known to be asymmetrically expressed in the LV versus RV myocardium, excepting those coding for beta-spectrin [4], ANKRD1 [9], BNP [6,9], calcium ATPase, matrix metalloproteinases, type 1 procollagens, and troponins [3]. In addition, other reports demonstrated that transcripts for proteins such as fibronectin, alpha-myosin heavy chain and transforming growth factor [26], and cytochrome c oxidase and heart isoforms of uncoupling proteins [27] are asymmetrically enriched in the LV versus RV mammalian myocardium.
Regulatory mechanisms resulting in LV/RV transcriptional differences in the newborn and early neonatal heart are largely unknown, but of special interest, because the functionally different roles of the two ventricles become apparent after birth. Our study characterizes the transcription status of the LV and RV at birth rather than the establishment of LV/RV transcription differences in the course of development [28]. In embryonic and fetal heart, expression of a number of transcription factors, including Hand1, Hand2, and Tbx5, shows LV/RV differences [29,30]. We found [9] that Hand1 and Hand2 are equally expressed in both the LV and RV of newborn piglets, suggesting that these factors are not involved in maintaining L/R ventricular transcriptional differences after birth.
In this work, among the genes whose expression levels differentiate between the LV and RV, there is a small cohort of genes which could be involved in concentric versus eccentric hypertrophy signalling (see Table 2). In this regard, several key regulators of muscle cell growth and proliferation (MAP3K7IP2, MSTN, PHB2, APOBEC3F) and gene expression (PTPLAD1, JMJD1C, CEP290) are differentially expressed between LV and RV piglet myocardium that may be relevant to intrinsic differences [31] that can regulate the chamber-dependent response of ventricular myocardium to workload. Interestingly, transition from "early" to "late" hypertension-induced hypertrophy in young adult rats is associated with predominant changes in expression of cell growth/proliferation and signal transduction factors [32].
Sequence analysis of the 32 cDNAs chosen based on differential LV/RV screening revealed a number of sequences, which may correspond to either previously uncharacterized genes or yet unidentified splice variants of the known cardioexpressed genes. In this sense, identification of the D36 fragment sequence (see Table 2) as being completely identical to that located within intron 8 of the pig ankrd1 gene led us to isolate and characterize three novel alternatively spliced ankrd1 variants which are predominantly expressed in the LV of neonatal and adult pig and human hearts and markedly upregulated in the ventricular myocardium at experimental heart failure [12]. Similarly, the D170 fragment (see Table 2), exhibiting homology with exon 1 and 3 sequences of the pig nppb gene, may represent a new form of alternative splicing of this cardioprotective factor.
Various cardiac disease states can result in an imbalance of chamber-associated expression patterns in ventricular myocardium. In the rat infarct model, a shift in chamberdependent gene expression towards relative downregulation of gene expression in the RV as compared to the LV has been reported [3]. In the porcine model of cardiotoxic cardiomyopathy we have demonstrated that the normal asymmetric LV/RV pattern of ANKRD1 mRNA and protein distribution was completely abolished at end-stage heart failure; improvement of cardiac performance resulted in the restoration of this gene's LV/RV asymmetric expression [9]. In the pig model of volume overload (eccentric hypertrophy promoting condition), angiotensinogen and preproendothelin expression levels were significantly upregulated in the RV while remaining uncharged in the LV [31]. In the mouse model of RV pressure-overload hypertrophy, over 10 transcripts showed significant upregulation in the afterload stressed RV, but not in the afterload stressed LV, including three genes from the Wnt signaling pathway, and genes involved in apoptosis [33]. In young rats, chronic hypoxia resulted in a shift from an LV-to an RV-predominant pattern in cytochrome c oxidase expression [27].
In sum, although not all of the identified genes with differential LV/RV expression have a clearly defined cardiacrelated function(s) at this time, the results of our work do advance the understanding of the complex mechanisms that could be involved in concentric versus eccentric remodeling of ventricular myocardium under normal conditions. More broadly, the identification of specific expression signatures of concentric versus eccentric hypertrophy may be useful in the elucidation of molecular pathways involved not only in physiological but also in pathological myocardial remodelling and heart failure.

Conclusions
Using an unbiased DDRT-PCR analysis, we were able to identify a set of genes with divergent LV versus RV expression. To our knowledge, this is the first study to account for large-scale gene expression profiling in early neonatal myocardium in mammals which revealed a certain molecular predisposition of the LV and RV, respectively, to concentric or eccentric hypertrophic remodeling. The reliability of these findings is supported by confirmation of the results by qRT-PCR and recognition of a fraction of the differentially expressed genes as known genes involved in pathological ventricular remodeling and heart failure. In addition, our data demonstrate chamber-dependent alterations in the expression of as yet uncharacterised novel genes that may be associated with different patterns of ventricular hypertrophic remodeling and can be used to study a board range of heart disease phenotypes.