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Am J Physiol Heart Circ Physiol 287: H1957-H1966, 2004. First published July 8, 2004; doi:10.1152/ajpheart.00568.2004
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Novel targets of ANG II regulation in mouse heart identified by serial analysis of gene expression

Faina Schwartz,2 Arvi Duka,1 Irena Duka,1 Jing Cui,1 and Haralambos Gavras1

1Department of Medicine, Hypertension and Atherosclerosis Section and 2Genetics Program, Boston University School of Medicine, Boston, Massachusetts 02118

Submitted 10 June 2004 ; accepted in final form 1 July 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although the central role of ANG II in cardiovascular homeostasis is well appreciated, the molecular circuitry of its many actions is not completely understood. With the use of serial analysis of gene expression to assess global transcriptional changes in the heart of mice after continuous 7-day ANG II administration, we identified patterns of gene expression indicative of cardiac remodeling, including coordinate regulation of genes previously described in a context of processes associated with hypertrophy and fibrosis. In addition, we discovered several novel ANG II targets, including characterized genes of known function, recently annotated genes of unknown function, and the putative genes not yet present in current databases. The serial analysis of gene expression approach to assess the role of ANG II presented in this report provides new venues for inquiries into ANG II-mediated cardiac function.

genome-wide transcriptional profiling; hypertension; cardiac remodeling; hypertrophy; fibrosis

THE KEY ROLE of ANG II in cardiovascular homeostasis is well documented. Although initially known for its vasoconstrictive action, the adverse effects of ANG II elevation on the heart independent of its hemodynamic influence were recognized in the early 1970s (7, 16), and the cardioprotective outcomes of the pharmacological interventions that either prevent the ANG II formation from the inactive angiotensinogen precursor (14) or inhibit its interaction with specific receptors (15, 48) are now well appreciated in clinical practice (6, 13, 17).

Experimental animal studies have shown that ANG II administration can cause multifocal myocardial lesions (16, 47), and, via ANG II type 1 receptors, directly induce cardiac remodeling that involves myocyte hypertrophy (9, 25), fibroblast proliferation, and subsequent fibrosis (47), thus leading to the development of pathological cardiac hypertrophy and, ultimately, heart failure. Both in vivo and in vitro studies revealed that the ANG II effects in the heart are accompanied by changes in gene expression in the cardiac myocytes, fibroblasts, and endothelial cells (24). While the queries of selected ANG II targets supplied important information on several molecular pathways underlying the physiological and pathophysiological actions of ANG II, an all-inclusive view is provided by the genome-wide transcriptional profiling that offers an opportunity to connect separate pieces of the puzzle through the identification of the new players not intuitively obvious from our present understanding of physiology. Thus simultaneous evaluation of 4,000 genes by the use of microarray technology to query the quantitative changes in left ventricular RNA in response to ANG II-induced progression, and, subsequently, regression of cardiac hypertrophy, revealed a set of genes previously not known to be associated with cardiac hypertrophy (12). Recently, a comprehensive analysis of the cardiac transcriptional response to acute and chronic ANG II treatments has been carried out using microarrays containing 22,000 unique transcript probes (26). One limitation of the microarray technology, however, is its dependence on the genes annotated to date. While the sequence of the human and mouse genomes is now available (22, 34, 50), the gene annotation is still incomplete, and the total number of genes, their respective transcripts, and RNA splicing variants, remains undetermined. Unlike microarrays, the serial analysis of gene expression (SAGE) method (49) does not rely on a priori gene knowledge and generates a catalogue of short sequences (tags) from all transcripts present in a given cell, tissue, or organ at the time of analysis. We used SAGE to compare global transcriptional profiling in the mouse heart subsequent to continuous 7-day ANG II administration with that of saline-infused animals. The quantitative changes in the heart transcriptome detected by SAGE are presented in this report.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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Animals. Male C57BL/6J mice weighing 25–27 g were purchased from Jackson Laboratory (Bar Harbor, ME). The animals were housed in the animal quarters, and all experiments were conducted in accordance with the "Guidelines for the Care and Use of Animals" approved by the Boston University School of Medicine. Continuous infusion of 0.9 µg/h ANG II (n = 6) or saline (n = 6) was carried out via subcutaneous osmotic minipumps (Alzet model 2001; Alza; Palo Alto, CA) for 7 days, as previously described (23, 41). Systolic blood pressure (BP) and heart rate measurements were obtained daily in conscious mice using a computerized, noninvasive tail-cuff system (model BP 2000, Visitech Systems). On day 7, the BP of ANG II-infused mice was 156.5 ± 12.9 mmHg (means ± SD), whereas the control mice remained normotensive (110.8 ± 9.5). After completion of BP measurements, the mice were euthanized with CO2, and the organs were harvested. The RNA prepared from the homogenized hearts of control and ANG II-treated mice was used to construct, respectively, C-7dH and ANG II-7dH SAGE libraries and for results verification by quantitative real-time PCR (Q-RT-PCR).

Mice administered ANG II (n = 8) and saline (n = 7) for 14 days were prepared using the same protocol. On day 14, the BP of ANG II-infused mice was 149.1 ± 14.8 mmHg, and the BP of control mice was 114.81 ± 6.95.

In the 6-h treatment group, the mean arterial pressure was recorded with a computerized data-acquisition system (Power Laboratory/400, ADInstruments) after direct catheterization of the iliac artery and connection of the arterial line to a BP transducer, as described elsewhere (23). At 6 h of ANG II (n = 7) or saline (n = 6) treatment, the mean arterial pressure values were, respectively, 134 ± 7.38 and 102.2 ± 9.15 mmHg.

After completion of the respective treatments, the total RNA was prepared from the heart, kidney, brain, and aorta of each animal for the Q-RT-PCR analysis presented in Fig. 2.



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  		Fig. 2. Q-RT-PCR expression analysis of novel ANG II-responsive genes. The y-axis shows the difference in the expression level of Hig1 (A), D930010J01Rik (B), 2900002H16Rik (C), and 2310008C07Rik (D) in the tissues from experimental relative to the control (baseline) animals, presented as (transcript copy number in experimental mice/transcript copy number in control mice) – 1. Q-RT-PCR, run in triplicate and including negative controls, were performed using total RNA from the brain, kidney, aorta, and heart of mice infused with ANG II (n = 6) or saline (n = 6) for 6 h (6 hr); ANG II (n = 6) or saline (n = 6) for 7 days (7D), and ANG II (n = 7) or saline (n = 8) for 14 days (14D). The heart salt sample shown in B was obtained from mice subjected to subtotal nephrectomy, as previously described (23, 29). After subtotal nephrectomy, experimental animals in this group were placed and maintained on 1% NaCl as drinking water for a maximal period of 35 days. Control mice were placed and maintained on regular drinking water. The average end-point blood pressure (BP) for the hypertensive animals used in this experiment (n = 6) was 153 ± 5 mmHg and for the control mice (n = 6) was 112 ± 7.5 mmHg. The transcript copy numbers for all samples were normalized with the data obtained from Gapdh endogenous control in the same tissue, and the means ± SE were calculated. Statistical analysis was performed by Student's t-test. Only statistically significant (P ≤ 0.05) differences in transcript abundance are shown.

 
Construction of SAGE libraries. Total RNA was prepared from homogenized hearts using TRIzol reagent according to the manufacturer's protocol (Invitrogen; Carlsbad, CA). To avoid potential contamination with genomic DNA, total RNA preparation was treated with DNase (Ambion; Austin, TX). To prepare SAGE libraries, ~25 µg of total heart RNA from the control (n = 6) and ANG II-infused (n = 6) animals were combined, and 100 µg of the pooled RNA were used to generate each library. Poly(A)+ RNA was isolated from the total RNA with oligo-(dT)25-coated magnetic beads (Dynal; Oslo, Norway). SAGE libraries were constructed as previously described (41) using NlaIII anchoring and BsmFI tagging restriction endonucleases (New England Biolabs; Beverly, MA) for tag generation. Concatemers were cloned in the SphI site of pZero (Invitrogen), and, following transformation of TOP10 Escherichia coli electrocompetent cells (Invitrogen), Zeocin-resistant colonies were checked for the presence of an insert by direct PCR using M13 forward and reverse primers. PCR-amplified inserts were sequenced at the Agencourt Bioscience (Beverly, MA). All electropherograms were checked manually to resolve sequencing artifacts and ambiguous base calls.

SAGE data analysis. Concatemer sequences of each clone were analyzed with the SAGE 2000 version 4.12 software (provided by Dr. Kenneth Kinzler's laboratory at Johns Hopkins Cancer Center, Baltimore, MD) to identify individual tags. Tags corresponding to linker sequences were discarded, and duplicate dimers were counted only once. The tag-to-gene assignment was carried out with the use of murine SAGE database at the National Center for Biotechnology Information at the site ftp.ncbi.nih.gov/pub/sage. Tags derived from mitochondrial transcripts were identified by sequence comparisons with the complete mouse mitochondrial genome (GenBank Accession No. J01420). Comparison between the two SAGE libraries was carried out using statistical functions available in the SAGE 2000 software for P value calculations and Monte Carlo simulations with a normalization value set to 18,000 tags per library and the minimal tag count setting of two for the two libraries combined. P ≤ 0.05 was considered significant. Assignment of tags corresponding to differentially expressed genes was individually checked with the use of the SAGEmap (http://www.ncbi.nlm.nih.gov/sage/) database, retrieving the corresponding mRNA or expressed sequence tag sequences from the UniGene database, and verifying the tag location within the sequence.

Q-RT-PCR. The TaqMan RT reagents (Applied Biosystems, Foster City, CA) were used to synthesize oligo-(dT)16-primed cDNA in a 50-µl reaction containing 1 µg of DNase I-treated total RNA, as previously described (41). Q-RT-PCR was performed with the ABI Prism 7900HT Sequence Detection System using a SYBR green-based protocol (Applied Biosystems). Oligonucleotide primers (Table 1) were designed with the Primer Express 2.0 software program (Applied Biosystems) and manufactured by Integrated DNA Technologies (Coralville, IA). All reactions were run in triplicate and included negative controls. The concentrations of the forward and reverse primers were between 300 and 900 nM. After initial denaturation at 95°C for 10 min, the cDNA products were amplified for 40 cycles consisting of denaturation at 95°C for 15 s and annealing and extension in a single step at 60°C for 1 min. The SDS version 2.1 software generated standard curves from 10-fold serial cDNA dilutions, and the threshold cycle was normalized for each standard curve. The range of slopes was between –3.22 and –4.01, where the value of –3.33 corresponds to 100% efficiency of the PCR. The copy numbers for all samples were normalized with the data obtained from the Gapdh endogenous control in the same tissue.


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  		Table 1. Primer pairs used in Q-RT-PCR analysis

 

   RESULTS
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 MATERIALS AND METHODS
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DISCUSSION
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The overall profiles of SAGE libraries. Using SAGE, we analyzed over 36,000 tags derived from transcripts present in the heart of C57Bl6 mice. Of these, 18,124 tags originated from the SAGE library prepared from the hearts of mice subjected to continuous 7-day ANG II administration (ANG II-7dH library), and 18,482 tags were derived from mice infused with saline (C-7dH control library). The number of individual transcripts identified in each library was, respectively, 6,609 and 6,433. A complete list of all SAGE tags from this study is available online at the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo) under Accession Nos. GSM23548 and GSM23547.

Comparison of the C-7dH and ANG II-7dH libraries revealed >100 tags significantly (P ≤ 0.05) differing in abundance. All tags listed in Table 2 for which the gene sequences are available have been individually checked to ascertain the presence and position of the tag within the corresponding gene. Whereas the majority of tags were localized at the last NlaIII (an anchoring enzyme used for the SAGE library construction) recognition site of the transcript, 14 tags (Table 2) were adjacent to a more upstream NlaIII site, suggesting potential alternative splicing or polyadenylation of the respective transcripts. However, the mitochondrially derived tags have not been subjected to the same analysis because the mitochondrial transcripts are polycistronic (46). Eleven tags shown in Table 2 matched more than one gene, and 19 tags remained unassigned. Of these, 13 (Table 2, underlined) have been previously detected in the SAGE libraries derived from the adult mouse heart (2) and cardiac fibroblasts (42).


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  		Table 2. Differentially expressed genes identified by SAGE

 
Because SAGE libraries were prepared from the pooled RNA samples, we assessed the degree of interanimal variability by examining a selection of differentially expressed genes by Q-RT-PCR using RNA from individual mice. Out of 32 randomly selected genes tested, 28 genes (82%) confirmed SAGE finding, with 24 genes (Fig. 1) reaching a statistical significance of P ≤ 0.05, as judged by Student's t-test.



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  		Fig. 1. Quantitative real-time PCR (Q-RT-PCR) verification of differential expression of a sample of genes identified by serial analysis of gene expression (SAGE). The y-axis shows the difference in the expression level of genes (listed in alphabetical order on the x-axis) in the hearts of mice administered ANG II for 7 days (n = 6) relative to the saline-infused controls (baseline) (n = 6), presented as (ANG II-7dH transcript copy number/C-7dH transcript copy number) – 1. Q-RT-PCR was performed using total RNA from the hearts of individual animals. The transcript copy numbers for all samples were normalized with the data obtained from Gapdh endogenous control, and the means ± SE were calculated. Statistical analysis was performed by Student's t-test, and the P values obtained for each gene are shown. The numbers in parentheses correspond to the numbers of tags detected in the C-7dH and ANG II-7dH SAGE libraries, respectively. Ckm, creatine kinase muscle; Crip1, cysteine-rich protein 1; Csrp1, cysteine and glycine-rich protein 1; Ddt, D-dopachrome tautomerase; Ech1, enoyl coenzyme A hydratase 1; Fn1, fibronectin 1; Gpx3, glutathione peroxidase; Hba-a1, hemoglobin-{alpha}, adult chain 1; Hig1, hypoxia-induced gene 1; Ldb3, LIM domain binding 3; Ly6e, lymphocyte antigen 6 locus E; Myo1g, myosin 1 G; Ndufa2, NADH dehydrogenase 1{alpha} subcomplex 2; Ppia, peptidylprolyl isomerase A; Serping1, serine proteinase inhibitor clade G, member 1; Sparc, secreted acidic cystein-rich glycoprotein; Uqcrfs1, ubiquinol cytochrome c reductase, Rieske iron-sulfur polypeptide 1.

 
Coordinate regulation of genes with related function. To evaluate the quantitative changes identified by SAGE in a physiological context, the transcripts have been grouped into functional categories defined by the Gene Ontology (http://www.geneontology.org) classification. As shown in Table 2, SAGE detected coordinate regulation of genes with related function within several categories, including energy metabolism, protein synthesis machinery, extracellular matrix (ECM), cytoskeletal organization and biogenesis, and calcium homeostasis. In addition, SAGE detected altered expression of genes for several transcription factors, intracellular signaling proteins, oxygen transporters, posttranslational processing proteins, and defense response. The modifications in the heart transcriptome revealed by SAGE are consistent with the quantitative and qualitative changes taking place during cardiac remodeling, myocyte hypertrophy, and fibrosis reported by others (27, 45). Importantly, several novel ANG II targets have been identified in this study.

Novel genes identified by SAGE with a likely function in ANG II-induced cardiovascular pathologies. In addition to genes with a designated Gene Ontology classification, we also detected six recently annotated genes and two expressed sequence tags, the functions of which have not yet been elucidated (Table 2). To begin our inquiry on the role of the novel genes in ANG II-mediated processes, we examined expression of Hig1, D930010J01Rik, 2900002H16Rik, and 2310008C07Rik in the heart, aorta, brain, and kidneys of control mice and mice administered ANG II for 6 h, 7 days, and 14 days. We found that, whereas each of the four genes has a unique expression profile (not shown), the response to ANG II is detected only in the cardiovascular system (Fig. 2).

Thus expression of Hig1, ubiquitously transcribed in the brain, kidney, heart, and aorta (not shown), changes in response to ANG II only in the heart and aorta (Fig. 2A). In the aorta, we detected upregulation of Hig1 in mice administered ANG II for 7 days, whereas in the heart, Hig1 is downregulated at days 7 and 14 of ANG II treatment (Fig. 2A).

Expression of D930010J01Rik, abundant in the heart, aorta, and the kidney, but barely detectable in the brain (not shown) changes in response to ANG II only in the heart. As shown in Fig. 2B, significant downregulation of D930010J01Rik is detected in the heart of mice after 7 days of continuous ANG II infusion. At day 14 of ANG II administration, the transcript level no longer differed from that of the controls. Unexpectedly, D930010J01Rik is also downregulated in mice fed a high-salt diet after subtotal nephrectomy, an established animal model of salt-sensitive hypertension (Fig. 2B).

The 2900002H16Rik gene is expressed in the heart, aorta, and brain, but not in the kidneys (not shown). However, differential expression in response to ANG II treatment was detected only in the heart (Fig. 2C). The 2900002H16Rik appears to be one of the early response genes because the change in its expression can be detected at 6 h of ANG II administration (Fig. 2C). Marked downregulation of 2900002H16Rik is seen on day 7 (Fig. 2C), but not on day 14 of infusion.

The 2310008C07Rik is prominently expressed in the heart and the aorta, but not in the brain or the kidneys (not shown), and a response to ANG II was observed only in the heart (Fig. 2D). In contrast to Hig1, D930010J01Rik, and 2900002H16Rik, which exhibited a decrease in transcript level on ANG II administration, expression of 2310008C07Rik is upregulated (Fig. 2D). Similarly to 2900002H16Rik, 2310008C07Rik appears to be an early response gene. The most pronounced change in expression is seen at 6 h of ANG II infusion, and is sustained at days 7 and 14 of treatment (Fig. 2D).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
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ANG II, an oligopeptide of eight amino acids, exerts several physiological influences on the cardiovascular system, affecting cardiac preload and stroke volume by regulating water and electrolyte homeostasis and vascular resistance and cardiac afterload by its vasoconstrictive action, stimulating the sympathetic nervous system at several levels, reducing the vagal activity, and promoting vascular and cardiac growth via direct interaction with ANG type 1 receptors (36). Not surprisingly, ANG II excess has a profound impact on cardiovascular homeostasis. In addition to hypertension, elevated ANG II directly induces cardiomyocyte hypertrophy and interstitial cardiac fibrosis, thus impairing myocyte contractility, oxygenation, and metabolism and leading to the progression to the pathological ventricular remodeling and heart failure (24, 27). Most of the ANG II-mediated effects in the heart occur through the activation of the ANG type 1 receptors (52), triggering a complex set of events that include stimulation of phospholipase C, subsequent activation of protein kinase C and release of Ca2+ from intracellular stores, phosphorylation of mitogen-activated protein kinases, transient induction of immediate-early genes, such as c-fos, c-myc, c-jun, and egr1, followed by transcriptional changes of the late response genes of the hypertrophic phase (24, 27). Despite great strides in the unraveling of the molecular circuitry underlying the development of cardiac hypertrophy and the transition to heart failure, our understanding of these processes remains incomplete. In this study, we used SAGE, a powerful global gene expression method, to identify novel cardiac ANG II targets.

With the use of SAGE to assess transcriptional profile of the mouse heart after continuous 7-day ANG II administration, we identified patterns of gene expression consistent with the remodeling process (45). Overall, our data are in agreement with those of previous reports emphasizing extensive reprogramming of the genes involved in ECM and cytoskeletal and sarcomere organization, the metabolic functions, calcium handling, and intracellular signaling, observed in experimental models of heart failure induced by pressure overload (32), genetic modifications (8, 32), or drug treatments (12, 25, 26), suggesting the existence of a common basic mechanism underlying the various aspects of cardiac remodeling in response to different initiating insults.

Increase in protein synthesis. Activation of protein synthesis is a hallmark of myocyte hypertrophy (27, 33), whereby the heart adapts to increased workload by augmenting its muscle mass through the enlargement of cell size in the absence of cell division (27). Consistent with cardiac hypertrophic response, SAGE shows prominent upregulation of the various components of the protein synthesis apparatus (Table 2), including structural constituents of small and large ribosomal subunits and translation initiation and elongation factors.

Repressed energy metabolism. The altered heart bioenergetics in response to ANG II is striking. Energy deficit is a well-recognized characteristic of the failing heart (45) and is known to reflect the depression of the mitochondrial function (37), impairment of the creatine kinase system (21), and defective lipid metabolism (53). Not surprisingly, SAGE showed coordinate downregulation of genes involved in ATP production, including the mitochondrial- and nuclear-encoded components of the oxidative phosphorylation, and creatine kinase. There is also a decrease in transcripts for proteins involved in lipid transport (fatty acid binding protein 3) and fatty acids {beta}-oxidation (Ech1, Hadhsc, Oxct, and Mlycd), whereas the sugar transporter gene (Dp1) is upregulated, consistent with the previously noted switch from lipid to glucose catabolism in the remodeling heart (45). The decline in the respiratory function also leads to increased production of reactive oxygen species in mitochondria (51), contributing to oxidative stress, and is potentially an important component of the ANG II-induced hypertension and cardiovascular disease (39). The upregulation of glutathione peroxidase 3 detected by SAGE (Table 2) likely reflects the cardiac response to oxidative stress.

ECM and cytoskeletal organization. Profound changes in the expression of genes related to cellular and extracellular architecture, previously highlighted in numerous studies of heart failure in the context of fibrosis, contractile dysfunction, and cytoskeletal stiffness, have been likewise detected by SAGE.

The changes indicative of myocardial fibrosis include both increased expression and altered composition of ECM components, such as upregulation of collagens type III and type XV, fibronectin Fn1, and Mglap (see Table 2); and upregulation of Sparc, the secreted acidic cysteine-rich glycoprotein (also known as osteonectin) (Table 2), a matricellular protein known to modulate cell shape, cell adhesion, and expression of ECM components (54). These genes are known targets of ANG II regulation (11, 18, 24, 25, 40, 45) and their upregulation in failing heart has been well documented (4, 12, 32, 43, 44).

The altered expression of myosin, recognized by SAGE, involves downregulation of {alpha}-myosin heavy chain, previously documented in hypertrophied human heart (28, 35, 44), and an upregulation of myosin 1G. A new ANG II target identified by SAGE is cardiomyopathy associated 4 (Cmya4) (also known as striated muscle UNC45), which encodes a protein with a role in sarcomere organization (38).

The changes in the cytoarchitecture suggestive of cardiac remodeling include modified expression of genes that encode both the structural components of the cytoskeleton and proteins that regulate cytoskeletal assembly. In particular, microtubular densification found in myocytes during cardiac hypertrophy and failure is thought to promote cardiac dysfunction by creating an increased load on myocytes, thus impeding sarcomere motion (20). Upregulation of {alpha}-tubulin detected by SAGE in this study has been previously reported in the failing human heart (44). We also identified an upregulation of the genes for actin monomer-binding proteins [protein tyrosine kinase 9-like (Ptk9l); or Twinfilin-2] and thymosin {beta}10 and differential regulation of four LIM-domain proteins [Csrp1, Csrp3, Crip1, and LIM domain binding 3 (Ldb3)] (Table 2) known to interact with the filamentous actin cross-linker {alpha}-actinin. Of these, Csrp1, Csrp3, and Crip1 are upregulated, whereas Ldb3 is downregulated. Csrp3, found exclusively in striated muscle, is known as a positive regulator of myogenesis with a function in cardiomyocyte cytoarchitecture (3). Mice with targeted disruption of this gene develop dilated cardiomyopathy with hypertrophy and heart failure (3). Its regulation by ANG II, to our knowledge, has not been reported. Ldb3 (also known as cypher) encodes another striated muscle-restricted protein and is likewise a newly recognized target of ANG II regulation. Ldb3 co-localizes with {alpha}-actinin-2 at Z lines in cardiac muscle and has been suggested to function as an adaptor between protein kinase C-mediated signaling and the cytoskeleton (55).

Intracellular signaling. One of the well-recognized indicators of cardiac hypertrophy is an upregulation of atrial natriuretic peptide (ANP) in ventricular myocytes (25, 30). The normal site of ANP synthesis in the adult mammalian heart is confined to the atria, whereas its expression in the ventricle is normally observed only during the fetal and neonate stages (30). In the adult, ventricular ANP expression signifies pathological conditions, such as hypertension and congestive heart failure. Interestingly, whereas increased expression of ANP in the whole heart has not been detected by SAGE in this study, SAGE identified upregulation of a recently annotated gene LOC230899, described in the GenBank database as a gene similar to ANP precursor (Table 2). Sequence analysis revealed that the LOC230899 gene pointed out by SAGE is a splicing variant of the ANP precursor message. Its relationship to the ANP transcript detected in the numerous studies of cardiac hypertrophy by Northern blot analysis, RT-PCR, or microarray methods (4, 8, 12, 25, 32, 43, 44) remains to be elucidated.

SAGE detected increased expression of the cardiac ankyrin repeat protein (Table 2), a cardiac-specific nuclear modulator of gene expression during cardiogenesis and a marker of cardiac hypertrophy in the adult (1). Its upregulation has been previously described in three distinct models of cardiac hypertrophy in the rat: constriction of the abdominal aorta, the spontaneously hypertensive rat, and the Dahl salt-sensitive rat (1).

Calcium homeostasis. SAGE shows downregulation of genes for calreticulin and phospholamban, proteins involved in Ca2+ handling. Calreticulin, a Ca2+-binding chaperone residing in the lumen of the endoplasmic reticulum, is known to affect intracellular Ca2+ homeostasis by modulating Ca2+ storage and transport (31). As an upstream regulator of calcineurin, the calcium-dependent protein phosphatase calreticulin plays a vital role in cardiac physiology and pathologies (31). Phospholamban, a sarcoplasmic reticulum membrane protein, is a regulator of SERCA2a, the cardiac isoform of sarco(endo)plasmic reticulum Ca2+-ATPase, diminished expression of which is considered to be one of the markers of chronic cardiac overload (19).

Transcription factors. SAGE revealed downregulation of two transcription factors (Table 2), zinc finger DHHC domain-containing 14 and homeodomain only protein, previously not known to be regulated by ANG II.

Genes of unknown function and unknown genes. An important advantage of SAGE over the other presently available high throughput methods of transcriptional profiling is its capability of detecting quantitative changes in the levels of unknown transcripts, including previously unrecognized genes as well as novel splicing variants (5). In the present study, we identified a difference in expression of several novel genes (Table 2). Whereas the sequences of six of these genes have been elucidated in a course of the mouse genome sequencing and annotation effort (10, 34), their functions remain unknown. The distinct expression profiles of the four novel genes in response to ANG II presented in this report (Fig. 2) suggest that each plays a unique role in ANG II-mediated cardiovascular effects.

In addition, SAGE revealed changes in the abundance of 19 tags (Table 2) that do not match sequences of known genes, 13 of which have been found in other SAGE libraries prepared from the adult mouse heart (2) and cardiac fibroblasts (42), thus underscoring the deficiencies in current gene databases. The isolation of transcripts corresponding to these unassigned tags is likely to fill in additional blanks in our present knowledge of the ANG II-mediated pathologies.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants P50-HL-55001 and U01-HL-66617.


   ACKNOWLEDGMENTS
 
We thank Dr. Kinzler for providing SAGE analysis software, and Michelle Liang for superb technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Schwartz, Dept. of Medicine, Genetics Program, Boston Univ. School of Medicine, 715 Albany St., L-320, Boston, MA 02118 (E-mail: fschw{at}bu.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Aihara Y, Kurabayashi M, Saito Y, Ohyama Y, Tanaka T, Takeda S, Tomaru K, Sekiguchi K, Arai M, Nakamura T, and Nagai R. Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter. Hypertension 36: 48–53, 2000.[Abstract/Free Full Text]
  2. Anisimov SV, Tarasov KV, Stern MD, Lakatta EG, and Boheler KR. A quantitative and validated SAGE transcriptome reference for adult mouse heart. Genomics 80: 213–222, 2002.[CrossRef][Web of Science][Medline]
  3. Arber S, Hunter JJ, Ross J Jr, Hongo M, Sansig G, Borg J, Perriard JC, Chien KR, and Caroni P. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell 88: 393–403, 1997.[CrossRef][Web of Science][Medline]
  4. Blaxall BC, Spang R, Rockman HA, and Koch WJ. Differential myocardial gene expression in the development and rescue of murine heart failure. Physiol Genomics 15: 105–114, 2003.[Abstract/Free Full Text]
  5. Boheler KR and Stern MD. The new role of SAGE in gene discovery. Trends Biotechnol 21: 55–57; discussion 57–58, 2003.[CrossRef][Web of Science][Medline]
  6. Brunner HR and Gavras H. Angiotensin blockade for hypertension: a promise fulfilled. Lancet 359: 990–992, 2002.[CrossRef][Web of Science][Medline]
  7. Brunner HR, Laragh JH, Baer L, Newton MA, Goodwin FT, Krakoff LR, Bard RH, and Buhler FR. Essential hypertension: renin and aldosterone, heart attack and stroke. N Engl J Med 286: 441–449, 1972.[Web of Science][Medline]
  8. Caron KM, James LR, Kim HS, Knowles J, Uhlir R, Mao L, Hagaman JR, Cascio W, Rockman H, and Smithies O. Cardiac hypertrophy and sudden death in mice with a genetically clamped renin transgene. Proc Natl Acad Sci USA 101: 3106–3111, 2004.[Abstract/Free Full Text]
  9. Dostal DE and Baker KM. Angiotensin II stimulation of left ventricular hypertrophy in adult rat heart. Mediation by the AT1 receptor. Am J Hypertens 5: 276–280, 1992.[Web of Science][Medline]
  10. FANTOM Consortium; RIKEN Genome Exploration Research Group. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature 420: 563–573, 2002.[CrossRef][Medline]
  11. Farhadian F, Contard F, Sabri A, Samuel JL, and Rappaport L. Fibronectin and basement membrane in cardiovascular organogenesis and disease pathogenesis. Cardiovasc Res 32: 433–442, 1996.[CrossRef][Web of Science][Medline]
  12. Friddle CJ, Koga T, Rubin EM, and Bristow J. Expression profiling reveals distinct sets of genes altered during induction and regression of cardiac hypertrophy. Proc Natl Acad Sci USA 97: 6745–6750, 2000.[Abstract/Free Full Text]
  13. Gavras H and Brunner HR. Role of angiotensin and its inhibition in hypertension, ischemic heart disease, and heart failure. Hypertension 37: 342–345, 2001.[Abstract/Free Full Text]
  14. Gavras H, Faxon DP, Berkoben J, Brunner HR, and Ryan TJ. Angiotensin converting enzyme inhibition in patients with congestive heart failure. Circulation 58: 770–776, 1978.[Abstract/Free Full Text]
  15. Gavras H, Flessas A, Ryan TJ, Brunner HR, Faxon DP, and Gavras I. Angiotensin II inhibition. Treatment of congestive cardiac failure in a high-renin hypertension. JAMA 238: 880–882, 1977.[Abstract/Free Full Text]
  16. Gavras H, Lever AF, Brown JJ, Macadam RF, and Robertson JI. Acute renal failure, tubular necrosis, and myocardial infarction induced in the rabbit by intravenous angiotensin II. Lancet 2: 19–22, 1971.[CrossRef][Web of Science][Medline]
  17. Gavras I and Gavras H. Angiotensin II as a cardiovascular risk factor. J Hum Hypertens 16, Suppl 2: S2-S6, 2002.
  18. Gonzalez A, Lopez B, Querejeta R, and Diez J. Regulation of myocardial fibrillar collagen by angiotensin II. A role in hypertensive heart disease? J Mol Cell Cardiol 34: 1585–1593, 2002.[CrossRef][Web of Science][Medline]
  19. Guo X, Chapman D, and Dhalla NS. Partial prevention of changes in SR gene expression in congestive heart failure due to myocardial infarction by enalapril or losartan. Mol Cell Biochem 254: 163–172, 2003.[CrossRef][Web of Science][Medline]
  20. Hein S, Kostin S, Heling A, Maeno Y, and Schaper J. The role of the cytoskeleton in heart failure. Cardiovasc Res 45: 273–278, 2000.[Abstract/Free Full Text]
  21. Ingwall JS, Kramer MF, Fifer MA, Lorell BH, Shemin R, Grossman W, and Allen PD. The creatine kinase system in normal and diseased human myocardium. N Engl J Med 313: 1050–1054, 1985.[Abstract]
  22. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 409: 860–921, 2001.[CrossRef][Medline]
  23. Johns C, Gavras I, Handy DE, Salomao A, and Gavras H. Models of experimental hypertension in mice. Hypertension 28: 1064–1069, 1996.[Abstract/Free Full Text]
  24. Kim S and Iwao H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev 52: 11–34, 2000.[Abstract/Free Full Text]
  25. Kim S, Ohta K, Hamaguchi A, Yukimura T, Miura K, and Iwao H. Angiotensin II induces cardiac phenotypic modulation and remodeling in vivo in rats. Hypertension 25: 1252–1259, 1995.[Abstract/Free Full Text]
  26. Larkin JE, Frank BC, Gaspard RM, Duka I, Gavras H, and Quackenbush J. Cardiac transcriptional response to acute and chronic angiotensin II treatments. Physiol Genomics 18: 152–166, 2004.[Abstract/Free Full Text]
  27. Lijnen P and Petrov V. Renin-angiotensin system, hypertrophy and gene expression in cardiac myocytes. J Mol Cell Cardiol 31: 949–970, 1999.[CrossRef][Web of Science][Medline]
  28. Lowes BD, Minobe W, Abraham WT, Rizeq MN, Bohlmeyer TJ, Quaife RA, Roden RL, Dutcher DL, Robertson AD, Voelkel NF, Badesch DB, Groves BM, Gilbert EM, and Bristow MR. Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium. J Clin Invest 100: 2315–2324, 1997.[Web of Science][Medline]
  29. Makaritsis KP, Handy DE, Johns C, Kobilka B, Gavras I, and Gavras H. Role of the alpha2B-adrenergic receptor in the development of salt-induced hypertension. Hypertension 33: 14–17, 1999.[Abstract/Free Full Text]
  30. McKenzie JC, Kelley KB, Merisko-Liversidge EM, Kennedy J, and Klein RM. Developmental pattern of ventricular atrial natriuretic peptide (ANP) expression in chronically hypoxic rats as an indicator of the hypertrophic process. J Mol Cell Cardiol 26: 753–767, 1994.[CrossRef][Web of Science][Medline]
  31. Michalak M, Lynch J, Groenendyk J, Guo L, Robert Parker JM, and Opas M. Calreticulin in cardiac development and pathology. Biochim Biophys Acta 1600: 32–37, 2002.[Medline]
  32. Mirotsou M, Watanabe CM, Schultz PG, Pratt RE, and Dzau VJ. Elucidating the molecular mechanism of cardiac remodeling using a comparative genomic approach. Physiol Genomics 15: 115–126, 2003.[Abstract/Free Full Text]
  33. Morkin E. Activation of synthetic processes in cardiac hypertrophy. Circ Res 35, Suppl II: 37–48, 1974.
  34. Mouse Genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse genome. Nature 420: 520–562, 2002.[CrossRef][Medline]
  35. Nakao K, Minobe W, Roden R, Bristow MR, and Leinwand LA. Myosin heavy chain gene expression in human heart failure. J Clin Invest 100: 2362–2370, 1997.[Web of Science][Medline]
  36. Nicholls MG and Robertson JI. The renin-angiotensin system in the year 2000. J Hum Hypertens 14: 649–666, 2000.[CrossRef][Web of Science][Medline]
  37. Peters TJ, Wells G, Oakley CM, Brooksby IA, Jenkins BS, Webb-Peploe MM, and Coltart DJ. Enzymic analysis of endomyocardial biopsy specimens from patients with cardiomyopathies. Br Heart J 39: 1333–1339, 1977.[Abstract/Free Full Text]
  38. Price MG, Landsverk ML, Barral JM, and Epstein HF. Two mammalian UNC-45 isoforms are related to distinct cytoskeletal and muscle-specific functions. J Cell Sci 115: 4013–4023, 2002.[Abstract/Free Full Text]
  39. Reckelhoff JF and Romero JC. Role of oxidative stress in angiotensin-induced hypertension. Am J Physiol Regul Integr Comp Physiol 284: R893–R912, 2003.[Abstract/Free Full Text]
  40. Schnee JM and Hsueh WA. Angiotensin II, adhesion, and cardiac fibrosis. Cardiovasc Res 46: 264–268, 2000.[Abstract/Free Full Text]
  41. Schwartz F, Duka A, Triantafyllidi E, Johns C, Duka I, Cui J, and Gavras H. Serial analysis of gene expression in mouse kidney following angiotensin II administration. Physiol Genomics 16: 90–98, 2003.[Abstract/Free Full Text]
  42. Smolenski A, Schultess J, Danielewski O, Garcia Arguinzonis MI, Thalheimer P, Kneitz S, Walter U, and Lohmann SM. Quantitative analysis of the cardiac fibroblast transcriptome-implications for NO/cGMP signaling. Genomics 83: 577–587, 2004.[CrossRef][Web of Science][Medline]
  43. Stanton LW, Garrard LJ, Damm D, Garrick BL, Lam A, Kapoun AM, Zheng Q, Protter AA, Schreiner GF, and White RT. Altered patterns of gene expression in response to myocardial infarction. Circ Res 86: 939–945, 2000.[Abstract/Free Full Text]
  44. Steenman M, Chen YW, Le Cunff M, Lamirault G, Varro A, Hoffman E, and Leger JJ. Transcriptomal analysis of failing and nonfailing human hearts. Physiol Genomics 12: 97–112, 2003.[Abstract/Free Full Text]
  45. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev 79: 215–262, 1999.[Abstract/Free Full Text]
  46. Taanman JW. The mitochondrial genome: structure, transcription, translation and replication. Biochim Biophys Acta 1410: 103–123, 1999.[Medline]
  47. Tan LB, Jalil JE, Pick R, Janicki JS, and Weber KT. Cardiac myocyte necrosis induced by angiotensin II. Circ Res 69: 1185–1195, 1991.[Abstract/Free Full Text]
  48. Turini GA, Brunner HR, Ferguson RK, Rivier JL, and Gavras H. Congestive heart failure in normotensive man. Haemodynamics, renin, and angiotensin II blockade. Br Heart J 40: 1134–1142, 1978.[Abstract/Free Full Text]
  49. Velculescu VE, Zhang L, Vogelstein B, and Kinzler KW. Serial analysis of gene expression. Science 270: 484–487, 1995.[Abstract/Free Full Text]
  50. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu-Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng Z, Di Francesco V, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan W, Ge W, Gong F, Gu Z, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke Z, Ketchum KA, Lai Z, Lei Y, Li Z, Li J, Liang Y, Lin X, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nusskern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun J, Wang Z, Wang A, Wang X, Wang J, Wei M, Wides R, Xiao C, Yan C, Yao A, Ye J, Zhan M, Zhang W, Zhang H, Zhao Q, Zheng L, Zhong F, Zhong W, Zhu S, Zhao S, Gilbert D, Baumhueter S, Spier G, Carter C, Cravchik A, Woodage T, Ali F, An H, Awe A, Baldwin D, Baden H, Barnstead M, Barrow I, Beeson K, Busam D, Carver A, Center A, Cheng ML, Curry L, Danaher S, Davenport L, Desilets R, Dietz S, Dodson K, Doup L, Ferriera S, Garg N, Gluecksmann A, Hart B, Haynes J, Haynes C, Heiner C, Hladun S, Hostin D, Houck J, Howland T, Ibegwam C, Johnson J, Kalush F, Kline L, Koduru S, Love A, Mann F, May D, McCawley S, McIntosh T, McMullen I, Moy M, Moy L, Murphy B, Nelson K, Pfannkoch C, Pratts E, Puri V, Qureshi H, Reardon M, Rodriguez R, Rogers YH, Romblad D, Ruhfel B, Scott R, Sitter C, Smallwood M, Stewart E, Strong R, Suh E, Thomas R, Tint NN, Tse S, Vech C, Wang G, Wetter J, Williams S, Williams M, Windsor S, Winn-Deen E, Wolfe K, Zaveri J, Zaveri K, Abril JF, Guigo R, Campbell MJ, Sjolander KV, Karlak B, Kejariwal A, Mi H, Lazareva B, Hatton T, Narechania A, Diemer K, Muruganujan A, Guo N, Sato S, Bafna V, Istrail S, Lippert R, Schwartz R, Walenz B, Yooseph S, Allen D, Basu A, Baxendale J, Blick L, Caminha M, Carnes-Stine J, Caulk P, Chiang YH, Coyne M, Dahlke C, Mays A, Dombroski M, Donnelly M, Ely D, Esparham S, Fosler C, Gire H, Glanowski S, Glasser K, Glodek A, Gorokhov M, Graham K, Gropman B, Harris M, Heil J, Henderson S, Hoover J, Jennings D, Jordan C, Jordan J, Kasha J, Kagan L, Kraft C, Levitsky A, Lewis M, Liu X, Lopez J, Ma D, Majoros W, McDaniel J, Murphy S, Newman M, Nguyen T, Nguyen N, Nodell M, Pan S, Peck J, Peterson M, Rowe W, Sanders R, Scott J, Simpson M, Smith T, Sprague A, Stockwell T, Turner R, Venter E, Wang M, Wen M, Wu D, Wu M, Xia A, Zandieh A, and Zhu X. The sequence of the human genome. Science 291: 1304–1351, 2001.[Abstract/Free Full Text]
  51. Wallace DC. A mitochondrial paradigm for degenerative diseases and ageing. Novartis Found Symp 235: 247–263, 2001.[Medline]
  52. Weir MR and Dzau VJ. The renin-angiotensin-aldosterone system: a specific target for hypertension management. Am J Hypertens 12: 205S-213S, 1999.[Web of Science][Medline]
  53. Wittels B and Spann JF Jr. Defective lipid metabolism in the failing heart. J Clin Invest 47: 1787–1794, 1968.[Web of Science][Medline]
  54. Yan Q and Sage EH. SPARC, a matricellular glycoprotein with important biological functions. J Histochem Cytochem 47: 1495–1506, 1999.[Free Full Text]
  55. Zhou Q, Ruiz-Lozano P, Martone ME, and Chen J. Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C. J Biol Chem 274: 19807–19813, 1999.[Abstract/Free Full Text]



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