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(Circulation. 2000;101:1990.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Application of cDNA Microarrays in Determining Molecular Phenotype in Cardiac Growth, Development, and Response to Injury

Patricia D. Sehl, MSc¹; Julie T. N. Tai, PhD¹; Kenneth J. Hillan, MD; Lesley A. Brown, PhD; Audrey Goddard, PhD; Renhui Yang, MD; Hongkui Jin, MD; David G. Lowe, PhD

From the Departments of Cardiovascular Research (P.D.S., J.T.N.T., L.A.B., R.Y., H.J., D.G.L.), Pathology (K.J.H), and Molecular Biology (A.G.), Genentech, Inc, South San Francisco, Calif.

Correspondence to David G. Lowe, PhD, Genentech, Inc, Cardiovascular Research, Mail Stop 42, 1 DNA Way, South San Francisco, CA 94080. E-mail lowe{at}gene.com

	Abstract

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Background—Normal myocardial development and the tissue response to cardiac stress are accompanied by marked changes in gene expression; however, the extent of these changes and their significance remain to be fully explored. We used cDNA microarrays for gene expression profiling in rat cardiac tissue samples to study developmental transitions and the response to myocardial infarction (MI).

Methods and Results—Microarrays with rat cDNAs for 86 known genes and 989 anonymous cDNAs obtained by molecular subtraction (representational difference analysis) of mRNA from sham-operated and 6-week post-MI samples were used in 2-color hybridization experiments. Twelve known genes previously associated with myocardial development were identified together with 10 uncharacterized expressed sequence tags and 36 genes not previously associated with cardiac development. After MI, genes associated with myocardial stress and wound healing exhibited differences in magnitude and expression kinetics, and 14 genes not previously associated with MI were identified. In situ hybridization revealed mRNA localization characteristic of wound healing and vascular and cardiomyocyte reactivity.

Conclusions—Tissue analysis of gene expression with cDNA microarrays provides a measure of transcriptional or posttranscriptional regulation and cellular recruitment. Our results demonstrate the complexity of gene regulation in the developing myocardium and show that cDNA microarrays can be used to monitor the evolution of the cardiac stress–inducible phenotype.

Key Words: genes • molecular biology • myocardial infarction

	Introduction

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Heart failure is a common outcome of hypertension or myocardial infarction (MI) and is a major contributor to cardiovascular morbidity and mortality. To compensate for the stress from an increased hemodynamic load, the heart undergoes compensatory hypertrophy, a response that restores lost function and normalizes wall stress, but hypertrophy is also an independent risk factor for heart failure.¹ The myocardial stress response is associated with changes in gene expression,² with the expression pattern reflecting the nature of the initial stress as well as the stage of compensatory hypertrophy or decompensated failure.^{3 4 5 6} One characteristic of this response is the reexpression of genes normally associated with cardiac development.^{7 8} Although not necessarily a direct correlation,^{4 9} the relation between pathological and developmental cardiac phenotypes remains to be fully explored.

The development of cDNA microarray technology to quantitatively monitor the expression of thousands of genes in parallel^{10 11 12} allows the detection of changes in the physiological status of tissues through changes in the molecular phenotype. These changes can reflect alterations in transcription, mRNA stability, or cellular composition in response to a changing environment.

In the present study, we used cDNA microarrays to analyze transcript levels in cardiac tissue samples. Our results provide a detailed view of the evolving phenotype of the developing heart and the myocardial response to stress.

	Methods

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Tissue Sources
The procedures used in the present study were approved by Genentech’s Institutional Animal Care and Use Committee. Rats were anesthetized with pentobarbital sodium, their hearts were excised, and left ventricle tissues were dissected and frozen in liquid nitrogen before storage at -70°C. Left and right ventricles from embryos at 13 days of gestation and from 1-day-old neonates were used in the developmental study. Ventricular tissue samples were pooled from 100 embryonic or 10 neonatal samples. For the post-MI study, coronary artery ligation was used to induce left ventricle infarction¹³ in 12-week-old male Sprague-Dawley rats. Sham surgeries were performed on the control groups for each time point. ECGs were used to document the development of infarcts according to the depth and persistence of pathological waves^{14 15} and to select animals with an infarct of at least 30%.^{16 17}

cDNA Amplification and Microarray Preparation
Eighty-six known genes (Table 1) were polymerase chain reaction (PCR)-amplified from rat heart cDNA by standard methods, cloned into pCR2.1 (Invitrogen), and sequenced for verification. Individual cDNA clones were PCR-amplified, purified, and prepared for microarray printing as described previously^{18 19} (details are available at http://cmgm.stanford/pbrown/index.html).

View this table: [in this window] [in a new window]   Table 1. Expression of Candidate Genes in Cardiac Development

View this table: [in this window] [in a new window]   Table 1A. Continued

Generation of Differentially Expressed cDNAs
cDNA fragments enriched for sequences induced or repressed after MI were prepared by representational difference analysis²⁰ with the use of modifications to the PCR-Select cDNA Subtraction Kit (Clontech). Briefly, first-strand cDNA was synthesized by using 2 µg of mRNA pooled from 3 sham-operated ventricles or three 6-week post-MI ventricles and 1 µmol/L of an oligo-dT primer with either RsaI or MslI restriction sites at the 5' end. After second-strand synthesis, the cDNA fragments were digested separately with RsaI or MslI, and the protocol was followed according to the manufacturer’s instructions. From the induced and repressed subtractions, 3744 clones were picked and screened with ³²P-labeled rat mitochondrial DNA²¹ to identify mitochondrial genomic fragments; 1963 (52%) nonmitochondrial clones were identified; and 989 clones were selected at random for microarray fabrication as described above.

Fluorescent Probes
Total RNA was prepared from tissues by use of RNA-Stat 60 (Tel-Test), and poly(A)⁺ RNA was isolated by using the PolyATract System (Promega). Reverse transcriptase (RT) reactions were carried out by using Superscript II RNase H^- RT (GIBCO) as described by the manufacturer, with the following modifications: 25 µL reactions contained 40 ng/µL poly(A)⁺ mRNA or 2 µg/µL total RNA, 80 ng/µL oligo dT primer, 1x Superscript first-strand buffer, 0.4 U/mL RNase Block (Stratagene), 500 µmol/L each of dATP, dGTP, and dTTP, 280 µmol/L dCTP (Boehringer-Mannheim), 40 µmol/L Cy5-dCTP or Cy3-dCTP (Amersham), and 8 U/µL Superscript II RT.

Data Analysis and Quantification
Hybridizations, washes, and array imaging were performed as previously described^{10 18 22} at Synteni Inc. Raw scores were normalized to the total fluorescence in either channel (Cy3 or Cy5), which is the sum of all fluorescence intensity values for the complete set of 1075 cDNAs. A ratio of differential gene expression was calculated from the normalized fluorescence intensity value obtained from each channel. Two different adult ventricular RNA samples were labeled with Cy3- and Cy5-dCTP and mixed in a reciprocal fashion to establish cutoff values for assigning differential expression. Deviation from an ideal ratio of 1 was found to range from 1.4 to 0.65. Because of this deviation, we chose a ratio of >1.5 for induction or <0.6 for repression. Expression ratios are reported as mean±SEM.

In Situ Hybridization
Frozen sections were prepared from rat ventricular tissue for in situ hybridization as previously described.²³ cDNA fragments were prepared for in vitro transcription²⁴ by PCR amplification with the use of gene-specific primers incorporating T3 and T7 RNA polymerase promoters.

	Results

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Developmental Gene Expression
Fluorescent cDNA prepared from 3 adult rat heart ventricles was pooled to provide an adult average against which each of three 13-day embryonic and 1-day-old neonatal samples were compared. In comparing embryonic and neonatal gene expression for the 86 known genes (Table 1), 46 cDNAs had a detectable fluorescent signal in embryonic RNA; there were 81 cDNAs with detectable signals in neonatal RNA. Relative to the adult, the fraction of genes that are differentially expressed was higher in the embryo (35 [76%] of 46) than in the neonate (18 [22%] of 81). Sixteen genes were differentially expressed in both samples, whereas 19 were differentially expressed only in the embryo (Table 1).

For the 989 anonymous cDNA clones on the array, we found differential expression for 117 (11.8%). After they were sequenced, these clones clustered to 48 genes modulated in either the embryonic or neonatal myocardium (Table 2). The majority of these (38 genes) were known, although they were not previously associated with cardiac development. In addition, we identified 10 cDNA clones corresponding to uncharacterized expressed sequence tags (ESTs) (Table 2).

View this table: [in this window] [in a new window]   Table 2. Identity and Expression of Genes in Cardiac Development

Gene Expression After MI
At 1, 3, 7, 14, 42, and 84 days after surgery, ventricular tissue samples were harvested from groups subjected to coronary artery ligation and from sham-operated groups (n=4 to 7 animals per group per time point). No hybridization signal was detected for cyclooxygenase 2, Bcl-2, and parathyroid hormone-like protein (PTHLP) in the set of known genes, whereas the remaining 83 genes (Table 1) gave a signal at the 6 time points. Of these, only 5 were induced for at least 1 time point after MI (Figure 1A and 1B). There is early elevation of mRNA encoding atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) at 1 day after MI, followed by further increases in ANP mRNA (Figure 1A). In contrast, BNP mRNA remains elevated at a constant level over the entire 12-week time course. The matrix proteins osteopontin, collagen III, and fibronectin each exhibit different kinetics of expression, but all returned to baseline within 6 weeks of surgery (Figure 1B).

View larger version (16K): [in this window] [in a new window]   Figure 1. Gene expression time course in post-MI ventricles. Data are expressed as ratio of expression between MI and sham-operated samples at 1 day (n=4), 3 days (n=5), 1 week (n=5), 2 weeks (n=5), 6 weeks (n=7), and 12 weeks (n=6) after surgery. Values are mean±SEM.

For the 989 unknown cDNAs, we found 63 clones exhibiting differential expression; these clones clustered to 14 different genes with elevated expression at 1 or more time points as long as 2 weeks after MI (Figure 1C, Table 3). No differential expression was observed for these genes at 6 or 12 weeks after MI.

View this table: [in this window] [in a new window]   Table 3. Time Course of Gene Expression After MI

mRNA Localization by In Situ Hybridization
Five genes were studied by in situ hybridization to determine the cellular source of differential expression. Hearts were harvested from sham-operated and MI rats at 4 and 10 days after surgery for analysis. Compared with normal myocardium (Figure 2A), infarcted myocardium revealed features typical of necrosis and hemorrhage (Figures 2B, 3A, and 3C). In normal heart, expression of vimentin (Figure 2C), p41-Arp2/3 protein complex subunit (ARC) (Figure 2D), and elongation factor-1 (EF-1, Figure 4A and 4B) was confined to vascular structures. Vimentin and EF-1 were expressed in blood vessel walls by smooth muscle cells and pericytes, whereas p41-ARC was expressed by endothelial cells lining small and large vessels as well as by vascular pericytes. The post-MI expression of each increased in spindle-shaped fibroblast-like cells within and adjacent to the region of damage (Figure 2E, 2F, and 2G). Expression of EF-1 also increased in blood vessels after MI (Figure 4). In contrast, cathepsin B was expressed in mononuclear phagocytes in infarcted hearts only (Figure 3A and 3B). Cathepsin B expression appeared higher at day 10 than at day 4, and its temporal and distinctive regional localization would be consistent with a role in organization and repair. Phospholemman was expressed at low levels in normal cardiac myocytes, with expression increasing after MI. The hybridization signal was higher in the ventricles and was highest in myocytes adjacent to the areas of damage (Figures 2H, 3C, and 3D).

View larger version (110K): [in this window] [in a new window]   Figure 2. Localization of EF-1, vimentin, and p41-ARC mRNA by in situ hybridization. Panel A shows low-power (x40) views of normal ventricular myocardium, and panel B shows corresponding area of MI, 4 days after ligation of left coronary artery. In panels A and B, myocardium (M), left ventricular chamber (LVC), necrotic myocardium (NM), blood vessel (V), and hemorrhage (H) are labeled. In normal heart, expression of vimentin (panel C) and p41-ARC (panel D) was confined to vascular structures (arrowheads). Panels E to H are dark-field images of serial sections from same sample as shown in panel B, 4 days after MI. Panels E, F, and G show in situ hybridization signals for EF-1, vimentin, and p41-ARC, respectively. Panel H shows expression of phospholemman in surviving myocytes.

View larger version (104K): [in this window] [in a new window]   Figure 3. Cathepsin B and phospholemman in situ hybridization. High-power (x200) images from areas of MI (day 10) are shown. Panels A and C are bright-field images, with corresponding dark-field images in panels B and D. Cathepsin B expression (A and B) was observed in macrophages adjacent to necrotic myocardium (arrowheads); phospholemman expression (C and D) was seen in surviving myocytes at sites of injury (arrowheads).

View larger version (106K): [in this window] [in a new window]   Figure 4. Vascular expression of EF-1 after MI. Panels A and C are bright-field images, with corresponding dark-field images in panels B and D (x200). EF-1 gene expression was low to undetectable in vessels of normal heart (arrowheads, A and B), whereas strong expression was observed in arterial smooth muscle/pericytes (C and D) after MI (day 10).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we applied cDNA microarray expression profiling to the study of developmental and pathological cardiac tissues. Confirmation of gene expression differences previously described during cardiac development (ANP and BNP,^{25 26} fibronectin and collagen III,⁹ sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase,²⁷ EF-1,²⁸ endothelin type A receptor,⁹ connexin40,²⁹ osteopontin,³⁰ manganese superoxide dismutase,⁹ troponin I,³¹ and heat-shock protein 22/-B crystallin³² ) or during the response to myocardial injury and stress (ANP and BNP,²⁵ osteopontin,^{30 33} collagen III,³⁴ and fibronectin³⁵ ) supports our identification of genes newly associated with these cardiac phenotypes. However, these results are not likely to be comprehensive even for the genes on our microarray. Signal dilution due to cell-type–specific expression or spatial regulation of mRNA levels could cause a signal below the differential expression threshold of detection. These considerations are particularly relevant to the heart, a complex arrangement of cardiomyocytes and endothelial, smooth muscle, endocardial, fibroblast, and neuroendocrine cells combined with spatial and regional specialization.

Developmental Gene Expression
Cardiac gene expression changes markedly in the progression from embryo to adult, reflecting the transition from a growing and developing organ to a stable adult heart. The largest number of differentially expressed mRNAs was detected when 13-day embryonic ventricles were compared with adult ventricles. Our data are consistent with results from EST cDNA sequencing, where clones more abundant in fetal and neonatal cDNA libraries frequently encoded regulatory and signal transduction proteins, reflecting a growing and less differentiated phenotype.³⁶

Reexpression of genes normally expressed in cardiac development has been observed in models of cardiac hypertrophy and failure.^{7 8 37} However, there is not a simple correlation between adaptive or pathological hypertrophy and developmental gene expression.^{4 9 14} Our data involving cardiac developmental gene expression provide a basis for more detailed testing of the relation between stress-inducible phenotypes in the adult myocardium and gene expression in myocardial development.

Expression Profiling in the Response to Injury
The rat model of surgical MI that we used is well characterized and represents an initial wound-healing response, followed by progression to cardiac dysfunction and failure.³⁸ The histological wound-healing response of the infarcted myocardium is reflected in the expression pattern of osteopontin,³³ fibronectin,³⁵ and collagen III,³⁴ which return to baseline expression levels between 2 and 6 weeks. In addition to gene expression reflecting the wound-healing response, ANP and BNP represent a cardiomyocyte response to stress,²⁵ with elevated expression as much as 12 weeks after MI. In situ localization of mRNA for 5 genes newly associated with MI highlights different cellular aspects of the tissue response to infarction, including wound healing in the infarcted region (vimentin,³⁹ EF-1,²⁸ and p41-ARC⁴⁰ ), infiltrating mononuclear phagocytes (cathepsin B⁴¹ ), vascular reactivity (p41-ARC⁴⁰ ), and myocyte reactivity (phospholemman⁴² ).

Our data on changes in gene expression after MI illustrate how microarrays may be used to monitor the progression of the tissue molecular phenotype and, as a discovery tool, to identify new genes associated with myocardial stress. This technology should be a potent hypothesis generator in experiments designed to elucidate the mechanisms of disease pathogenesis and to determine the basis of drug action.

Study Limitations
The microarray that we designed was intended to evaluate the expression of known and unknown genes by comparing developmental gene expression with the response to MI. In fact, very few differentially expressed genes were detected in the post-MI time course. This is particularly striking given the extent of differential expression identified in the embryo and neonate compared with the adult by use of the same microarrays. We consider several possibilities. First, the relation between the myocardial stress phenotype and developmental gene expression^{7 8 37} may, in fact, be quite limited, a conclusion supported by previous studies using fewer candidate genes.^{4 5 9}

Alternatively, there are still extensive changes in gene expression after MI that are below the level of microarray sensitivity. This limitation may be overcome by isolating selected regions of the myocardium to enrich for expression differences, by examining isolated cell populations, or by technically improving assay precision and reproducibility. The latter case is illustrated by our results with cathepsin B: multiple cDNAs were on the arrays, but varying numbers of clones were hybridized at or above the differential expression threshold (Tables 2 and 3). Beyond 2 weeks after MI, no cathepsin B differential expression was detected. However, the cathepsin B cDNAs were obtained by molecular subtraction of mRNA of sham-operated and 6-week post-MI myocardium. Enrichment for cathepsin B cDNAs, as reflected in the large number of randomly picked cathepsin B cDNAs on the microarray, clearly suggests that there is elevated expression at later times. Although these results suggest that molecular subtraction by representation difference analysis does provide enrichment, improvements in sensitivity will be required to evaluate the full advantage of this strategy. However, when all expressed sequences are known as a result of the genome projects, the molecular subtraction approach will not be a necessary antecedent to array fabrication.

Distinguishing among closely related members of a gene family is also problematic with microarrays, inasmuch as we were not able to detect expected changes in actin and myosin gene expression.^{43 44} This is clearly an area requiring technical improvement with modified hybridization conditions or the use of shorter DNA fragments/oligonucleotides.

In summary, the present study establishes the utility of cDNA microarrays to monitor the expression profile of a tissue over time. In addition to function, anatomy, and histology, expression analysis by microarray can be expected to become an integral part of studying the response of the heart to a changing environment. Additional work on myocardial expression profiling can be expected to help unravel the relation between gene expression and cardiac function in the normal and diseased heart.

	Acknowledgments

 
The authors wish to thank Aldona Kallok for assistance in manuscript preparation.

	Footnotes

 
¹ P.D. Sehl and Dr Tai contributed equally to this study.

Received March 22, 1999; revision received October 25, 1999; accepted November 18, 1999.

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