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Am J Physiol Heart Circ Physiol 294: H2268-H2275, 2008. First published March 14, 2008; doi:10.1152/ajpheart.91494.2007
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Transcription repression and blocks in cell cycle progression in hypoplastic left heart syndrome

Katheryn Gambetta,1 Mohamad K. Al-Ahdab,1 Michel N. Ilbawi,1,2 Nahidh Hassaniya,3 and Madhu Gupta1,2

1The Heart Institute for Children, Hope Children's Hospital, Oak Lawn; and 2Department of Pediatrics, Rush University Medical Center, Chicago, Illinois; and 3Loma Linda University, Loma Linda, California

Submitted 19 December 2007 ; accepted in final form 7 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Hypoplastic left heart syndrome (HLHS) is characterized by abnormally developed atrial septum and a severe underdevelopment of the left side of the heart. Despite significant advances in its surgical management, little is known about the molecular abnormalities in this syndrome. To gain molecular insights into HLHS, expression profiling by gene-chip microarray (Affymetrix U133 2.0) and by real-time RT-PCR was performed in the atrial septum of patients diagnosed with HLHS and compared with age-matched non-HLHS patients. Hierarchical clustering of all expressed genes with a P < 0.01 of all tissue samples showed two main clusters, one of HLHS and the other of non-HLHS, suggesting different expression patterns by the two groups. Net affix followed by real-time RT-PCR analysis identified the differentially expressed genes to be those involved in chromatin remodeling, cell cycle regulation, and transcriptional regulation. These included remodeling factors, histone deactylase 2 and SET and MYND domain containing 1; transcription factors, FoxP1, and components of the calcineurin-nuclear factor of activated T cells signaling pathway; and cell cycle regulators, cyclin-dependent kinase (CDK)-4, phosphatase and tensin homolog, and p18. Since these factors play essential roles in heart growth and development, the abnormal expression pattern suggests that these molecules may contribute to the pathogenesis of HLHS.

FoxP1; MYND domain containing transcription factor; phosphatase and tensin homolog; calcineurin; nuclear factor of activated T cells

HYPOPLASTIC left heart syndrome (HLHS) is a relatively common and complex cardiac malformation that is most therapeutically challenging. It is characterized by a marked underdevelopment of the left side of the heart, which includes the small left atrium, small or absent left ventricle (LV), stenosis or atresia of mitral valve, hypoplasia or atresia of aortic valve, and severe hypoplasia of ascending aorta. The atrial septum is also abnormal, resulting in varying degrees of interatrial communication ranging from restrictive pinhole-size to nonrestrictive foramen ovale. It is often associated with an underdeveloped Eustachian valve, which normally directs placental return to the left heart chambers during fetal life (7). Fetal echocardiography usually demonstrates atrial septal defect in a more cephalad location than that of the normal patent foramen ovale. The right atrium, right ventricle, and pulmonary arteries are grossly dilated, and a widely patent ductus arteriosus is present. At the cellular level, the heart shows disorganized muscle bundles, small myocytes with scant cytoplasm, and abundant connective tissues (4). HLHS accounts for 7.5% of all congenital heart disease diagnosed in the first year of life (11). Before surgical treatment became available, this malformation was universally fatal with 90% of deaths occurring in the first month of life (14).

The etiology or the molecular abnormalities in HLHS remain unknown. Although a clear linkage of genetic factors to HLHS has yet to be established, a high incidence of cardiac defects in the first degree relatives, an autosomal recessive mode of inheritance, and familial clustering as well as chromosomal abnormalities such as trisomies 13 and 18, Turner's syndrome, and terminal deletion of 11q23-qter have been reported (17, 35, 37). These reports collectively suggest a possible role of genetic factors in HLHS. The objective of the present study is to provide a comparative expression analysis of HLHS and non-HLHS hearts by using gene-chip microarray and real-time RT-PCR. Our data show an altered expression of chromatin remodeling factors, transcription factors, FoxP1, and components of calcineurin-nuclear factor of activated T cells signaling, suggesting an overall transcriptional repression in HLHS. In addition, we found an altered expression of key cell cycle regulators, which point to a block in cell cycle progression.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Affymetrix human genome array (HG-U133 Plus 2.0) was used to analyze the expression pattern of the atrial septum in HLHS and non-HLHS patients. This microarray features 1,300,000 unique oligonucleotides, which encompass 47,000 transcripts and variants, representing ~39,000 human genes.

Tissue collection. This protocol was approved by the Research Ethics Committee of Advocate Christ Medical Center and adheres to the principles of the Declaration of Helsinki as well as to the Title 45, US code of Federal Regulation, Part 46, Protection of Human Subjects (Revised November 2001 and effective December 2001). Informed consent was obtained from the parents of all patients before the collection of tissue samples. The tissues were excised and immediately snap frozen in liquid nitrogen and stored at –80°C.

Atrial septal tissues were excised and collected by the same surgeon (M. N. Ilbawi) shortly after starting cardiopulmonary bypass (bypass duration, 75 ± 15 min) during open-heart surgery. Preoperative systemic arterial saturations were comparable in the two groups of patients. In the HLHS group, they ranged from 70% to 85%; in the non-HLHS group, they ranged from 75% to 85%. The majority of patients in both groups received PGE1 treatment before tissue collection. All patients were screened for chromosomal abnormalities, and those who tested negative were included in the study.

Characteristics of HLHS group. A total of 14 patients fulfilling the criteria of true HLHS proposed by the Congenital Heart Surgery Nomenclature and Database Project in 2000 (42) were included in the study. All variant forms of HLHS were excluded. Atrial septal tissues were collected within 3 wk of life (5–21 days) at the time of the classic Norwood procedure or Norwood procedure with Sano modification. The mitral valve-to-LV apex dimension was <1 cm, and in many cases the LV was visually nonexistent.

Characteristics of non-HLHS group. A total of 17 age-matched patients (2–23 days) with cardiac lesions other than HLHS were used as controls. The LV size in this group of patients was within the normal range. The mitral valve-to-LV apex dimension was >3 cm in all subjects. Atrial septal biopsies were collected during open-heart corrective surgery.

Age, sex, cardiac lesions, LV dimensions, aortic root dimensions, and any medications at the time of tissue collection in the HLHS and non-HLHS groups are summarized in Table 1.


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  		Table 1. Characteristics of HLHS and non-HLHS patients

 
RNA extraction. Total RNA was extracted using TRIzol reagent (GIBCO, Rockville, MD) according to the manufacturer's recommendations. Residual genomic DNA was digested with RNAase free DNAase (Qiagen, Valencia, CA) at room temperature for 30 min. The integrity of the purified RNA was tested by a bioanalyzer, and only the samples demonstrating a 28S-to-18S ratio of 1.8 to 2.0 were included in the study.

Microarray processing. cDNA was prepared according to the protocols provided with the Affymetrix U133 GeneChip system (Affymetrix, Santa Clara, CA). Five micrograms of total RNA were used in the first-strand cDNA synthesis using Superscript III (GIBCO, Gaithersburg, MD) and T7-d(T)24 primer GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-[dT]24. The second-strand cDNA synthesis was performed at 16°C using Escherichia coli DNA ligase, DNA polymerase I, and RNase H, followed by T4 DNA polymerase to blunt the ends of newly synthesized cDNA. The cDNA was purified through phenol-chloroform and ethanol precipitation. The purified cDNA was converted to biotin-labeled cRNA by using a BioArray High Yield RNA Transcript labeling kit (Enzo Diagnostics, Farmingdale, NY) in an in vitro transcription reaction for 5 h at 37°C.

Microarray hybridization. cRNA was fragmented by incubating in a buffer containing (in mmol/l) 200 Tris-acetate, 500 KOAc, and 150 MgOAc for 35 min at 94°C. Fifteen micrograms of fragmented cRNA were mixed with eukaryotic hybridization controls (containing control cRNA and oligonucleotide B2) and hybridized with a preequilibrated HG-U133 Plus 2.0 Affymetrix chip for 16 h at 45°C. The chips were washed in a fluidic station with low-stringency buffer containing 6x standard saline phosphate with EDTA, 0.01% Tween 20, and 0.005% antifoam for 10 cycles and with high-stringency buffer containing 100 mmol/l N-morpholino-ethanesulfonic acid, 0.1 mol/l NaCl, and 0.01% Tween 20 for 4 cycles and then stained with streptavidin phycoerythrin. This process was followed by incubation with normal goat immunoglobulin G and biotinylated mouse anti-streptavidin antibody and restaining with streptavidin phycoerythrin.

Statistical analysis of microarray data. The chips were scanned in a HP G2500A ChipScanner (Affymetrix) to detect hybridization signals. Image output files were visually examined for major chip defects and hybridization artifacts, and raw data were processed with GeneChip Microarray Analysis Suite 5.0 software (Affymetrix). The image from each chip was scaled such that the absolute signal intensities were adjusted to target intensity and reported as a nonnegative numerical quantity. Data were imported into SAS version 7 (SAS, Cary, NC) for further analysis. Probe sets with significant intensity differences between HLHS and non-HLHS were identified by applying Wilcoxon signed-rank test to the absolute signal intensities of each gene. The use of a nonparametric, paired Wilcoxon test was selected over that of a standard t-test to avoid a distributional assumption; the threshold was set at a P value of 0.01 or less. By the use of this P value, the number of genes that could have reached significance by chance alone was determined and compared with the number of observed significant genes, and a signal-to-noise ratio was computed.

Real-time quantitative PCR. Primer sequences were selected for each target gene containing minimal internal structures (i.e., hairpins and primer dimers) and optimum melting temperatures (i.e., melting temperatures each within 1°C of the other) determined using the primer express software (Applied Biosystems, Foster City, CA). The specificity of each primer for each gene was tested with standard nucleotide basic local alignment search tool (BLASTn) covered on the National Center of Biotechnology Information homepage (http://www.ncbi.nlm.nih.gov). Table 2 depicts the primer sequences used for real-time PCR. The primers were tested for their ability to produce a single product of correct size upon agarose gel analysis. Each sample was analyzed in triplicate for every primer pair. To correct for variations of RNA amounts and cDNA synthesis efficacy, GAPDH was used as a housekeeping gene, since β-actin mRNA appeared to be unsuitable for normalizing gene expression in patients with congenital heart defects (19).


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  		Table 2. Nucleotide sequence of the primer pairs utilized for real-time RT-PCR

 
Thermal cycling and SYBRgreen fluorescence detection were done in a real-time PCR thermal cycler (TaqMan, ABI PRISM 7900 sequence detection system; Applied Biosystems). The 10-µl reactions were prepared in 384-well plates (Applied Biosystems) as follows: 5 µl of SYBRgreen qPCR reaction mix, 2 µM each of forward and reverse primers, and 1.0 ng of cDNA. Thermal cycling conditions used were 2 min at 50°C and 5 min at 95°C followed by 40 cycles of 30 s at 95°C and 30 s at 60°C. Melting curve analysis was added after the final PCR cycle to evaluate the presence of nonspecific PCR products and primer dimers. During the initial optimization runs, fivefold serial dilutions were employed to demonstrate a linearity of amplification range for each gene.

The threshold cycle value represents the PCR cycle at which an increase in SYBRgreen fluorescence above a baseline signal can be first detected. For each sample, the amount of mRNA was quantified by real-time PCR relative to 1 ng of total RNA used in a RT reaction. A no-RT reaction was included with each sample to exclude the possibility of genomic contamination. Serial dilutions of control templates were used to calculate the amplification efficiencies of each primer pair. A total of six to eight hearts were analyzed each from non-HLHS control and HLHS groups, and each analysis was performed in triplicate with at least two different RT reactions of each sample. Expression ratios were calculated using the Pfaffl method, and the data were presented as normalized expression ratios of control and HLHS groups and expressed as means ± SD.

Statistical analysis. Values from HLHS and non-HLHS patients were compared using a Student's t-test.


   RESULTS AND DISCUSSION
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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Heart development, a complex and dynamic process, is regulated in a precise manner by transcription factors and signaling pathways involved in cell proliferation, cell migration, and muscle cell differentiation. Such precision does not appear to be achieved by the strict regulation of a single pathway in and of itself but rather through a vast network of interacting and cross-regulating pathways that, by their sheer complexity, provide for a system of checks and balances that fine-tunes the behavior of cells. In this study, we took a global approach to identify genes demonstrating an abnormal expression in HLHS. We compared the expression profile of the atrial septum of HLHS and non-HLHS patients by using gene-chip microarray. This technology has been previously used to understand disease processes in several organs including the heart (8, 39). Since it was not possible to obtain age-matched normal heart samples, we used non-HLHS hearts as controls. To avoid bias against a particular defect, a heterogeneous group of non-HLHS hearts were used for comparison. The only consistent differentiating feature between the HLHS and non-HLHS patients was the absence of left heart hypoplasia in the latter.

Differential gene expression profiles among HLHS and non-HLHS populations. To examine the expression patterns in HLHS and non-HLHS patients, all genes with significant expression (P < 0.01) in all samples were subjected to hierarchical cluster analysis. Data presented in Fig. 1 show clear-cut differences in the expression profile of the HLHS and non-HLHS groups. A total of 288 genes with known functions showed differential expression; of these, 130 genes were overexpressed and 158 were underexpressed in the HLHS group. These genes were further evaluated by choosing a minimum of 1.5-fold change in the expression level between the two groups. Table 3 is a list of such genes that were considered to be differentially expressed in HLHS. A functional analysis using Net Affix and Spotfire softwares revealed that the identified differentially expressed genes play important roles in cell cycle regulation, gene transcription, and cytoskeletal organization. The expression of some of these genes was also verified by real-time PCR.


Figure 1
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  		Fig. 1. Hierarchical clustering of differentially expressed genes in atrial septal tissue of hypoplastic left heart syndrome (HLHS) and non-HLHS patients. Each bar represents a pool of 2 patient samples, and each row represents a single gene. Based on the range of color, the level of gene expression can be visualized. The color scale represents bright green as –2.9-fold change to bright red as 3.2-fold change with the overall range of 6.1-fold differences.

 

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  		Table 3. List of differentially expressed genes in HLHS

 
Cell cycle regulation in HLHS. The development of the heart from cardiac progenitors to a fully functional four-chambered structure requires many rounds of myocyte cell division, which is most active in the embryonic heart and continues even during early postnatal life (30). With maturation, the proliferative capacity of myocytes declines and then ceases (20, 38). During each round of division, the cells complete an ordered series of events collectively known as a cell cycle. This cycle includes an accurate duplication of the genome during the DNA synthesis phase (S phase) and the segregation of complete sets of chromosomes to each of the daughter cells in the M phase. The somatic cell cycle also contains Gap phases known as G1, which connects the completion of the M phase to the initiation of the S phase in the next cycle, and G2, which separates the S and M phases. Each phase of a cell cycle is tightly regulated by the temporal activation and inactivation of several cell cycle regulatory proteins belonging to three main groups of proteins: cell division cycle 2/cyclin-dependent kinases (CDK2, CDK4, CDK6, and Cdc2), cyclins (A, B, D, and E), and CDK inhibitory proteins (p15, p16, p18, p19, p21, p27 kip1, and p57 kip2). The binding of cyclins to CDKs results in CDK activation, which catalyzes events required for cell cycle initiation and transitions. CDK activity is inhibited by CDKIs or by phosphorylation mediated by Wee1 and Myt1 (23, 24).

Microarray analysis of the HLHS hearts demonstrated an altered expression of several cell cycle regulators, which mainly suggest blocks in cell cycle progression. Fold change in the expression level of these regulators and their cellular targets and functions is summarized in Tables 3 and 4. HLHS hearts showed an increased expression of several inhibitors of cell cycle progression. This included WEE1, RNA binding motif single stranded interacting protein 1 (RBMS1), CDK inhibitors that interfere with the transition of G1 to the S phase such as p18, p21, and p57 kip2, and KH domain containing RNA binding signal transduction associated 1 (KHDRBS1) (24, 43). The increased expression of homeodomain interacting protein kinase 2 (HIPK2), sterile alpha motif and leucine zipper containing kinase (ZAK), and phosphatase and tensin homolog (PTEN), a tumor suppressor that induces cell cycle arrest, inhibits cell growth, and causes apoptotic cell death (31), was likewise noted. In contrast, there was a reduced expression of regulators involved in cell cycle progression in HLHS hearts. This included Rad17, a promoter of the cell cycle and a component of checkpoint signaling cascade involved in DNA repair to maintain genome stability, and CDK-4 (Table 4 and Fig. 2), which enhances cell cycle progression by promoting cell entry from G0 to G1 (9, 16, 34). The expression of other CDKs or cyclins was not altered in HLHS. Taken together, our data indicate that HLHS hearts demonstrate blocks at several steps of cell cycle progression, mediated by an upregulation of cell cycle inhibitory proteins and by a downregulation of CDK-4 and Rad17. Protein degradation through ubiquitin-mediated proteolysis has been shown to play an important role in cell cycle regulation (3). The HLHS hearts showed an increased expression of several ring finger proteins with ubiquitin ligase activity, suggesting that increased ubiquitination may also be playing a role in HLHS. Ring finger proteins demonstrating altered expression in HLHS are listed in Table 3.


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  		Table 4. List of genes involved in cell-cycle regulation with altered expression in HLHS

 

Figure 2
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  		Fig. 2. Real-time RT-PCR analysis of various factors in atrial tissue samples of controls and HLHS patients. Each bar represents fold change in cycle threshold (CT) values normalized for GAPDH expression compared with control values that have been taken as 100. Values are from 6 to 8 hearts of each group (see MATERIALS AND METHODS) and expressed as means ± SD. p18, cyclin-dependent kinase (CDK) inhibitory protein; PTEN, phosphatase and tensin homolog; NFATC3, nuclear factor of activated T cell 3; FoxP, forkhead box protein; SMYD1, SET and MYND domain containing transcription factor.

 
Chromatin remodeling factors in HLHS. Chromatin structure imposes significant obstacles on all aspects of transcription that are mediated by RNA polymerase II. Recent studies have demonstrated that histone modification through acetylation and deacetylation plays an important role in controlling cardiac growth and gene expression in response to acute and chronic stress (2). Table 3 lists chromatin modifiers that showed an abnormal expression in HLHS as identified by microarray analysis. Figure 2 shows the fold change in the expression levels of some of these modifiers as assessed by real-time RT-PCR. The HLHS hearts showed an increased expression of histone deactylase 2 (HDAC2), SET and MYND domain containing transcription factor (SMYD1), and COOH-terminal binding protein 2 (CtBP2). They also showed a downregulation of high-mobility group box 1 (HMGB1), which is involved in maintaining chromatin structure (21).

SET and MYND domains are known to mediate distinct chromatin modifications, where MYND domain recruits HDACs for deacetylation and SET domain possesses histone-methyl transferase activity for methylating deactylated residues, thereby causing a more permanent state of chromatin silencing (27). Recent studies have shown that Bop, a SET and MYND-containing transcriptional repressor, is dependent on histone deacetylase for its repressive activity, particularly in cardiac and skeletal muscle precursors as well as in cardiomyocytes of chick and mouse embryos (12). The increased expression of both SMYD1 and HDAC2 in HLHS hearts is highly suggestive of transcription repression in HLHS.

CtBP2 belongs to a family of the CtBP family of proteins that predominantly function as transcriptional corepressors (6, 41). CtBP2 has been found to repress transcription through several mechanisms including the recruitment of HDAC, through its interaction with friends of GATA (FOG) coregulatory proteins, and with polycomb group protein Polycomb 2 (36), as well as by a direct repression of gene transcription (42). CtBP2 null mutant mice reveal defects in cardiac morphogenesis (15). We observed an increased expression of CtBP2 as well as another repressor, cut-1CAAT displacement protein, which represses gene transcription via the recruitment of histone deacetylase activity (1, 28). Our data thus suggest that transcriptional silencing may occur in HLHS, furthering the possibility that by inhibiting gene transcription, the developmental maturation of cardiac myocytes might be affected.

Transcription factors in HLHS. Many transcription factors arbitrate specific events during cardiogenesis (22). In this study, we observed the neonatal atrial septum to express several transcription factors that have been shown to play important regulatory roles in various aspects of cardiac morphogenesis. These include dHAND, T-box (TBX) family members (TBX1–TBX3 and TBX5), Pitx2, and GATA family members (GATA4–GATA6). However, the expression level of these factors was similar in HLHS and non-HLHS hearts. Interestingly, HLHS hearts demonstrated a reduced expression of FoxP1, a finding also confirmed by real-time RT-PCR.

FoxP1 is a transcriptional repressor that belongs to a subfamily of genes containing a homologous DNA binding domain called forkhead that binds to a consensus sequence found in the promoters and enhancers of many genes. Recent studies have elucidated the importance of FoxP1 in cardiovascular development (44). An inactivation of FoxP1 in mice leads to endocardial cushion defects, outflow tract obstruction, and ventricular septal defects. Defects in cardiomyocyte proliferation and maturation also result in a thinning of ventricular walls in these mice (44). Thus a reduced expression of FoxP1, as observed in the present study, suggests its possible contributory role in HLHS.

We also observed a decreased expression of components of the nuclear factor of activated T cell (NFATC)/calcineurin signaling pathway in HLHS. NFATC3 belongs to the family of NFATC, which forms a component of the intracellular calcium signaling pathway that regulates gene transcription. Calcineurin is a Ca/calmodulin-dependent protein phosphatase, which dephosphorylates NFATC family members (NFATC1–NFATC4) during periods of sustained elevation in calcium. The binding of calcineurin to NFATC facilitates the NFATC translocation to the nucleus. Once inside the nucleus, NFATC binds to its consensus DNA sites in its target genes to control gene transcription. Upon termination of the calcium signal, NFATC gets rephosphorylated by GSK-3β, resulting in its translocation back to the cytoplasm. This pathway has been shown to play an important role in cardiac development. In mouse embryos by E11.5, the expression of NFATC1 becomes restricted to cells lining the outflow tract and heart valves (10), and NFATC3 mutant embryos display occluded outflow tract and underdeveloped aortic, pulmonary, mitral, and tricuspid valves, accompanied by small and hypertrophied ventricular chambers (32). We observed a reduced expression of both NFATC3 and calcineurin in HLHS hearts (Fig. 2 and Table. 3), suggesting the contribution of this pathway to the left heart underdevelopment. This is also suggested by the altered expression of several other proteins involved in maintaining calcium homeostasis inside the cell. These included phospholamaban (PLN), calmodulin binding protein, striatin 3 (STRN3), myozenin/calcarin-1, and inositol triphosphate receptor-1 (Table 3). We also observed a decreased expression of Sp3, which is known to regulate the expression of calcium-handling proteins such as sarcoplasmic calcium ATPase (5).

ECM and cytoskeleton proteins in HLHS. There are conflicting data regarding collagen matrix in HLHS; some studies show increased collagen expression (4) and others reduced collagen expression (33). In our study, we found no difference in the collagen expression in HLHS compared with non-HLHS hearts. We observed an increased expression of cytoskeletal proteins such as actinin alpha 2 (ACTN2), an actin and calcium binding protein, nebulette (NEBL), which is predominantly expressed in the fetal heart, and cofilin, which is also an actin binding protein (25, 26). There was a reduced expression of cell adhesion molecules tenacin C, necessary for endocardial cushion formation during cardiac development (29), and of plakophilin, which plays a role in the assembly of junctional proteins. The null mutation of plakophilin in transgenic mice results in reduced trabeculation and a disarray of the cytoskeleton with impaired cell-to-cell contacts (13). HLHS hearts also demonstrated a decreased expression of several genes that regulate microtubule polymerization that included dynein, tubulin delta 1, and microtubule associated protein 4. Our data thus collectively suggest that there is altered expression of cytoskeletal proteins in HLHS.

Conclusion

In summary, our study has provided a comprehensive gene-expression analysis of the atrial septum of HLHS and non-HLHS patients by microarray and identified components of some of the biological pathways that are affected in HLHS. The altered expression of these important regulatory proteins was confirmed by real-time RT-PCR. Our findings suggest a defect in chromatin remodeling, cell cycle regulation, and transcriptional regulation in HLHS. Although many of these genes have been studied extensively in mice during cardiac development, ours is the first to demonstrate the altered expression of these genes in the atrial tissue of children born with HLHS. It may serve as a foundation for further studies probing for regional differences, e.g., the left versus right heart, as well as for changes during the earlier stages of heart development.

However, it should be reemphasized that our study constituted a comparative analysis of the gene expression profiles primarily of the septum primum component of the atrial septum of neonates with HLHS or non-HLHS. Normal heart tissues were not used as controls. Hence, the abnormal findings observed in HLHS hearts realistically represented differences from those of the non-HLHS hearts. The non-HLHS group of patients comprised a heterogeneous group of cardiac malformation where hypoplasia of the left atrium and left heart structures was not a feature. Since the expression of the reported molecules did not differ among the members of the non-HLHS group, it can be assumed that these findings are related to the underlying abnormalities in HLHS. Whether the observed molecular changes in the atrial septum represent a global developmental defect of the rest of the left heart structures will be a challenge to future investigations. Our findings suggest that the altered expression of these molecules may be responsible for the abnormal atrial septum formation and support a hemodynamic concept of reduced left atrial filling during early development, which serves to inhibit the progression of left heart growth during normal fetal life.


   ACKNOWLEDGMENTS
 
We thank Dr. Rene A. Arcilla for advice and support of this work. We also thank Jason Monroe (Northwestern University, Chicago, IL) for the processing of the gene chips and Yianna Kazakos for technical assistance and sample collection.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Gupta, Dept. of Pediatrics, Rush Univ. Medical Center, 1653 W. Congress Pkwy, Chicago, IL 60612 (e-mail: gupta{at}thic.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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