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1 Departments of Pediatrics
2 Neuroscience, Albert Einstein College of Medicine, Bronx, New York
3 Department of Pharmaceutical Sciences, Idaho State University College of Pharmacy, Pocatello, Idaho
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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and -4E (eIF-2 and eIF-4E) was found in CCH, whereas eIF-4E was downregulated in 1- and 2-wk CIH, suggesting that eIF-4E is likely to play an important role in the cardiac hypertrophy observed in CCH-treated mice. On the other hand, the specific downregulation of heart development-related genes (e.g., notch gene homolog-1, MAD homolog-4) and the upregulation of proteolysis genes (e.g., calpain-5) in the CIH heart can explain the lack of hypertrophy in CIH. Interestingly, apoptosis was enhanced in CCH but not CIH, and this was correlated with an upregulation of proapoptotic genes and downregulation of anti-apoptotic genes in CCH. In summary, our results indicate that 1) the pattern of gene response to CCH is different from that of CIH in mouse heart, and 2) the identified expression differences in certain gene groups are helpful in dissecting mechanisms responsible for phenotypes observed. patterns of hypoxia; cardiac hypertrophy; cDNA microarray; apoptosis; initiation factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Technologies like microarrays have not yet been used to study differences between CCH and CIH in heart. Because 1) there has been a paucity of studies on the effect of chronic hypoxia on heart in early life, which may be different from that in the adult, and 2) it is possible that CCH and CIH have a different impact on the heart, we performed transcriptomic analyses and compared differences in gene expression between these two types of hypoxia using the neonatal mouse.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Histology.
A total of nine hearts (3 hearts/group) from animals exposed to CCH, CIH, or room air for 4 wk were obtained for histological examination. Fresh hearts were fixed in 4% paraformaldehyde overnight and transferred to 75% ethanol with double-distilled H2O for paraffin embedding. The sections were stained with hematoxylin and eosin. The sizes of cardiomyocytes were measured as transverse areas (µm2) of the cells in at least 10 fields of sections (x400 magnitude) using the image AxioVision 4.1 software (Zeiss, Thornwood, NY).
Microarrays.
Arrays were hybridized with cDNA from four individual animals at each age (1, 2, or 4 wk) and treatment (CCH, CIH, or normoxia), as shown in Supplemental Fig. S1A (available at the Physiological Genomics web site).1
The slides (28,704 spots, representing 7,455 distinct genes with known protein products in http://genome-www5.stanford.edu/cgi-bin/source//sourceBatchSearch, with several spotted sequences probing the same gene), 11,686 expressed sequence tags (ESTs) whose annotation was incomplete at the date of the study (eliminated from the expression analysis), and 192 bacterial sequences for quality control of the arrays were obtained from the Microarray Facility of AECOM. The hybridization process was performed according to the instructions of the core facility. Briefly, total RNA (60 µg), extracted with TRIzol (Invitrogen), was used to synthesize a fluorescently labeled cDNA probe by direct incorporation with either Cy3 or Cy5 fluorescent dye (Amersham Biosciences) in separate reactions. Fluorescent cDNA probes were prehybridized with blocking solution for 1 h before being applied to pretreated and prehybridized microarray slides. Hybridization was done in GeneMachines HybChamber and incubated overnight at 50°C. After incubation, each slide was washed to remove unbound cDNAs and SDS, dried, and scanned with a GenePix 4100A scanner (Axon Instruments) at 600 V (635 nm) and 550 V (532 nm).
We adopted an experimental strategy (experimental design and flow chart in Supplemental Fig. S1A) similar to that used in previous studies (3). This strategy was termed "multiple yellow" (MYS), since most spots on the hybridized slide should appear yellow in an 8-bit pseudocolor image (example in Supplemental Fig. S1B). As presented in the DISCUSSION section and in the Supplemental Material, MYS provides a similar detection accuracy of the regulated genes compared with the widely used dye swapping (DSS) and reference sample (RSS) strategies (16) but has a considerable advantage in cost and flexibility. Each slide was hybridized with heart cDNA obtained from a male mouse (labeled with Cy5) and a female mouse (labeled with Cy3), both of which were subjected to the same treatment for the same period of time. Thus all comparisons between hypoxia and normoxia used animals of the same gender composition.
Images were acquired and primarily analyzed with GenePix Pro 4.1 software. The background-subtracted signals were normalized with an in-house developed iterative algorithm similar to those used in previous publications (12, 13), alternating within-array normalization and interarray normalization until the average-corrected ratio differed by <5% from the previous one (14). Individual measurements of genes for all 12 mice studied in each period (1, 2, or 4 wk) were further divided by the average of the corresponding normoxic values, and then the results for each group of four mice (i.e., normoxic, CCH, and CIH) were rescaled with respect to the average of that group. The ratios obtained by proportioning the normalized green and red fluorescence intensities of a spot with hypoxic cDNAs to the normalized green and red fluorescence intensities of a matched spot with corresponding normoxic cDNAs were averaged for both channels. In the case of a gene probed in multiple spots, the expression ratio was the weighted average ratios, as previously described (12). Detection of significantly regulated genes relied on both fold changes in expression ratio (limited by the technical noise of the method and expression variability among animals) and the statistical significance of the two-tailed t-test with a Bonferroni-type adjustment applied to the redundancy groups (14). The data set (series no. GSE2271) was deposited in the Gene Expression Omnibus (GEO) database: http://www.ncbi.nlm.nih.gov/geo/. Profiling of the data was accomplished using hierarchical clustering algorithm, with the software available from http://rana.lbl.gov/index.htm.
Quantitative real-time RT-PCR.
The two-step quantitative real-time RT-PCR (QRT-PCR) SYBR Green method (Applied Biosystems) was used to compare and confirm the levels of selected interesting genes. Primers were devised with the software Primer 3 and synthesized at Invitrogen. The cDNA synthesis and QRT-PCR were done according to previously described methods (29). Relative ratios of fluorescent intensities of products from hypoxia to normoxia were calculated by using the 2–
Ct method, where Ct is cycle threshold (17), and ß-actin amplicons were used as loading control. Specific primers are listed in Supplemental Table S5.
Western blotting.
Total protein was prepared using buffer as previously described (1). The concentration of protein lysates was determined with the bicinchoninic acid protein assay kit. Protein samples (20 µg) were isolated through SDS-PAGE electrophoresis using 10% Novex Bis-Tris gel and then electrophoretically transferred to a polyvinylidene difluoride membrane. Nonspecific binding sites were blocked, and the membranes were incubated overnight at 4°C with primary antibodies [eukaryotic translation initiation factor (eIF)-4E and eIF-4E (Ser209) from Cell Signaling, eIF-2 and eIF-2 (Ser52) from Abcam, and internal control Hsc70 from Stressgen]. The signals were visualized by incubating with horseradish peroxidase (HRP)-conjugated secondary antibody followed by enhanced chemiluminescence. Band densities were quantified using the Personal Densitometer SI scanner (Molecular Dynamics, Sunnyvale, CA) and analyzed with the aid of ImageQuaNT image analysis software (Molecular Dynamics).
Apoptosis detection.
In situ terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay (Roche Applied Science) was used to detect apoptotic nuclei and quantified as percentage of apoptotic nuclei per total nuclei. Sections were first deparaffinized and rehydrated, and then the manufacture's instructions were followed. Briefly, sections were stripped of protein by incubation with pepsin (0.25%, pH 2.0) for 15–20 min at 37°C. For positive control, a section of normoxic control heart was treated with DNase I to produce artificially fragmented nuclear DNA. Samples were incubated with TUNEL reaction mix for 60 min at 37°C in a dark, humidified chamber. Total nuclei were counterstained with DAPI. Samples were first observed under a fluorescence microscope, then treated with anti-fluorescein antibody conjugated with alkaline phosphatase (AP), and observed under a light microscope. Nuclei were counted using the software AxioVision 4.1. Results are expressed as mean values ± SD. Differences in means were considered statistically significant if P < 0.05, using unpaired Student's t-test.
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RESULTS |
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4 wk of age in CCH mice (Fig. 1A). Heart weight and size increased significantly in CCH animals but remained unchanged in CIH compared with normoxic controls (Fig. 1B and Fig. 2A). This increased heart weight in CCH mice was significant after 1 wk in hypoxia, and this difference continued to be pronounced at 2 and 4 wk of age (Fig. 1B). A similar pattern was also detected in total protein/heart in CCH mice but not CIH mice (Fig. 2F). The ratio of heart weight to body weight increased in both CCH and CIH, but the difference from control was greater in CCH (Fig. 1C). Hematocrit increased in both CCH and CIH, but the difference was more significant in CCH at all ages (Fig. 1D).
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Overview of gene expression using cDNA microarray.
Our results showed that a substantial number of genes have altered their expression in the hearts of both CCH- and CIH-treated mice. Both individual variability and reproducibility of gene expression pattern of mice subjected to the same treatment are illustrated in Fig. 4A, Supplemental Table S6, and Supplemental Fig. S4. We found that a total of 549 genes were upregulated and 375 genes downregulated in CCH heart (Fig. 3A). A substantial number of genes were also altered in CIH, but the majority were downregulated: 294 genes upregulated and 440 genes downregulated (Fig. 3B). At 1, 2, and 4 wk with CCH, there were 272, 856, and 294 upregulated genes and 110, 613, and 303 downregulated genes, respectively. Likewise, with CIH there were 375, 440, and 150 upregulated and 440, 795, and 68 downregulated genes at these same time points. Remarkably, in both treatments, the largest number of altered genes was after 2 wk of exposure to hypoxia. Genes that altered their expression at all three time points are listed in Supplemental Tables S1 and S2.
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The experimental design allowed us to compare gene expression in the two genders. Fig. 3E presents the fold-change difference between male and female mice subjected to 1 wk of CIH in the entire set of four mice. We found no difference in the type of regulation between the two genders (all differences <50% fold change) and no significant bias of fold change toward one gender or another (symmetrical distribution of differences).
Similarities in gene expression between CCH and CIH.
During chronic hypoxia, whether CCH or CIH, some of the regulated genes responded qualitatively in a similar fashion in the heart (Table 3). These included stress-responding genes (e.g., heat shock and redox genes), genes involved in vascular dilation, angiogenesis, and heme biosynthesis. For example, the gene that encodes a thioredoxin-interacting protein inhibits the function of thioredoxin; therefore, downregulation of this gene by 2.3-fold in CCH and 1.6-fold in CIH suggests enhancement of antioxidant function. A recent report has shown that a downregulation of this gene is involved in cardiac hypertrophy (28). The gene EGL nine homolog 1, which is involved in the degradation of the protein of hypoxia-inducible factor (HIF), was upregulated by 5-fold in CCH and 2.4-fold in CIH, whereas EGL nine homolog 3 was downregulated by 1.7-fold in CCH and 1.8-fold in CIH (7).
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Divergent transcriptomic effects of CCH and CIH.
Some gene families were differently altered in the two conditions. These genes are likely to be involved in inducing distinct phenotypes between CCH and CIH hearts. For example, genes encoding eukaryotic initiation factors and genes encoding ribosomal protein subunits were mostly upregulated in CCH and downregulated in CIH. We identified a total of 23 eIFs that were regulated by hypoxia in the mouse heart, with more upregulated genes in CCH and more downregulated genes in CIH hearts. This may explain the increased protein synthesis in CCH heart and subsequent myocardial hypertrophy (Fig. 2F and Fig. 4A). Indeed, previous studies have indicated that eIFs and their phosphorylation are important in cardiac hypertrophy (4). CCH and CIH induced similar regulation of genes such as eIF3s, eIF4g2, and eIF4el3 but opposite regulations of genes such as eIF3s10, eIF3s2, and eIF2c2. To determine whether eIF proteins increase and possibly play a role in cardiac hypertrophy, eIF-2 and eIF-4E were studied in this work. Western blotting showed that both eIF-2 and eIF-4E increased 1.5-fold at 1 wk in CCH. We also showed that phosphorylated eIF-4E (Ser209) increased by 1.8- to 2.0-fold at 1 and 2 wk in CCH, and this increase was more remarkable than the increase in total protein level of eIF-4E. The changes of total as well as phosphorylated eIF-2 and eIF-4E in CIH heart were not significant. The gene eIF-4E, along with the upregulation of eIF-4E binding protein 2, an inhibitor of eIF-4E, control the translation efficiency and are likely to be important in cardiac hypertrophy in CCH (4).
The divergent effects of CCH and CIH on heart gene expression were also observed when apoptotic and Rho/MAPK signaling genes were considered. For example, most of the proapoptotic genes were upregulated and most of the anti-apoptotic genes were downregulated in CCH but not in CIH (Fig. 5, A and B, and Table 4). This suggested that myocardial apoptosis might be enhanced in the CCH model. To further test this hypothesis, TUNEL staining was performed in both CCH and CIH heart sections. At least 20 consecutive high-magnification images were captured from each section of CCH, CIH, or control hearts. The ratio of apoptotic nuclei to total nuclei was significantly higher in the heart after 4 wk of CCH treatment (0.86%) compared with the age-matched normoxic controls (0.34%, P < 0.05; Fig. 5, C and E). No significant difference was found in the heart samples after 4 wk of CIH treatment (0.44%, P > 0.05; Fig. 5, C and F). This result correlated well with the changes in proapoptotic gene as well as anti-apoptotic gene expression in CCH and CIH. Furthermore, some genes, the function of which is related to either the Rho pathway or MAPK pathway, were differentially regulated in CCH and CIH hearts. Most members related to the Rho pathway were upregulated in CCH, but all were downregulated in CIH (Supplemental Fig. S2C); most of the altered MAPK pathway-related genes were upregulated in CCH but not in CIH (Supplemental Fig. S2D).
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1.5- to 2.3-fold in CCH but downregulated by a similar magnitude in CIH. The homeodomain-interacting protein kinase 1, a suppressor of homeodomain transcription factor, which is involved also in development, was downregulated by 2.2-fold in CCH but upregulated by 1.6- to 2.2-fold in CIH. In addition, the upregulation of GATA-2 in CCH but its downregulation in CIH may explain the different effects of CCH and CIH on cardiac muscle size (19). Furthermore, the small optic lobes homolog gene, which contains a calpain domain, was upregulated 7.0-fold in CIH but downregulated 5.8-fold in CCH. This suggests that the small optic lobe gene may be important in hypoxia-reoxygenation-induced injury or proteolysis (26). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the current study, several clusters of genes that are related to certain specific biological processes were significantly altered by the hypoxia treatments. One of the altered gene clusters is related to the translational initiation factors in CCH and CIH. In CCH, genes encoding eIFs as well as ribosomal proteins were mostly upregulated, as measured by microarrays and QRT-PCR as well as by Western blot analysis [eIF-2, eIF-2 (Ser52), eIF-4E, and eIF-4E (Ser209)]. Upregulation of these genes and their proteins enhances protein synthesis. Protein levels and phosphorylated proteins of eIFs may also have an effect on translation and protein synthesis. While the relation between phosphorylated eIF-2 and protein synthesis may not be well understood, that of phosphorylated eIF-4E is well known. For example, Tuxworth et al. (25) found that eIF-4E phosphorylation and protein synthesis are increased concomitantly in response to stimuli that induce hypertrophic growth in adult cardiocytes (25). This is consistent with our in vivo results: both eIF-4E protein level and phosphorylated eIF-4E (Ser209) increased in CCH after 1 and 2 wk, an increase that is expected to promote protein synthesis. In CIH, eIF-4E was downregulated at both 1 and 2 wk, a condition that explains the absence of cardiac hypertrophy. Therefore, we raise the distinct hypothesis that the enhanced protein synthesis machinery (via eIFs) plays an important role in the hypertrophy of heart in CCH. The eIF RNA and protein results and the hypothesis of increased protein synthesis in CCH are further supported by our other data showing increased cell size of cardiac myocytes as well as increased total protein (Fig. 2F).
Signaling pathways that induce hypertrophy and enlargement of heart size include two gene families: the Rho GTPases and the MAPKs. Because 1) several members of Rho GTPases have been reported to be involved in cardiac hypertrophy (11), and 2) two members of the Rho GTPases (Arhgap10 and Arhgap18) were upregulated in CCH, we believe that such pathways actually contributed in inducing cardiac hypertrophy in CCH. Indeed, most members of MAPK have been identified in our work to be upregulated in CCH but not in CIH. Such changes may be related to increased heart mass in CCH (24). Combined with other results from our microarray study, such as the downregulation of thioredoxin-interacting protein and upregulation of GATA-2, which are already known to be involved in cardiac hypertrophy (28, 19), we believe that hypertrophy of cardiac myocytes in CCH is the result of coordinated regulation on expression of various gene families.
Of great interest is the fact that the increase in protein synthesis in the heart in hypoxia contrasts to the decrease in protein synthesis in most organs (such as brain and kidney; Supplemental Fig. S3, A and B). The question of how different is protein synthesis in the hypoxic heart compared with other organs is intriguing. We have indeed alluded to this difference in our previous work (18). Interestingly, the lungs also increase in weight or at least do not reduce their weights in hypoxia, as do the kidneys and to a lesser degree the brain (Supplemental Fig. S3, C and D, and unpublished observations), suggesting that the heart and lungs behave in a similar manner and enhance protein synthesis for adaptation to the hypoxic stress. Although muscle fiber stretching such as in hypertension or overload can induce cardiac hypertrophy, we believe that hypoxia directly induces the hypertrophy. This partly agrees with in vitro studies showing that mild hypoxia (10% O2) induces hypertrophy of cardiomyocytes of rat (15).
Because hypoxia can change cell fate, we further asked whether programmed cell death takes place, especially because we have evidence that, in CCH heart, the genes involved in apoptosis are regulated. In situ TUNEL staining confirmed that changes in gene expression paralleled those in apoptosis. This result further supports the notion that, during CCH, the heart undergoes remodeling that is not restricted only to hypertrophy. There is indeed a more complicated process that induces apoptosis (9, 27).
Although the increase in cardiac and cell size in CIH was not impressive, we did additional experiments to determine whether a more severe hypoxia in the intermittent model (7.5% O2 instead of 11% O2) would induce a hypertrophy similar to CCH. With this more severe paradigm, the heart and cell size were even much smaller than in controls, suggesting that the lack of hypertrophy in CIH is due to the nature of this particular stress model. Downregulation of most subunits of mitochondrial complex I in CIH but not in CCH suggested possible mitochondrial functional inhibition and a resultant shortage of ATP supply in the organ (10). Along with downregulation of several genes involved in cardiac development (Supplemental Figs. S2, E and G), these may constitute the underlying molecular mechanisms in CIH.
In conclusion, our results show that CCH and CIH have different impacts on heart phenotype and that the respective genetic responses provide a molecular basis for these phenotypic differences. In CCH, the heart is characterized by a robust right ventricular hypertrophy and larger cardiac mass. This phenotype creates an imbalance with the continuous relative shortage of O2 supply and with an induction of proapoptotic genes, which may constitute a major mechanism for heart failure. By contrast, in CIH, mitochondrial dysfunction and cardiac growth inhibition in early life may be more important.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. G. Haddad, Dept. of Pediatrics and Neuroscience, Albert Einstein College of Medicine, Rose F. Kennedy Center 845A, 1410 Pelham Parkway South, Bronx, NY 10461 (e-mail: ghaddad{at}aecom.yu.edu).
10.1152/physiolgenomics.00217.2004
1 The Supplemental Material for this article (Supplemental Figs. S1–S4 and Supplemental Tables S1–S6) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00217.2004/DC1. 
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P < 0.01 compared with CIH.
