Gene expression profile of mouse myocardium with transgenic overexpression of A1 adenosine receptors

Amy R. Lankford1, Anne M. Byford1, Kevin J. Ashton4, Brent A. French2, Jae K. Lee3, John P. Headrick3 and G. Paul Matherne1

1 Department of Pediatrics and Cardiovascular Research Center, University of Virginia Health System, Charlottesville Virginia 22908
2 Department of Biomedical Engineering, University of Virginia Health System, Charlottesville Virginia 22908
3 Health Evaluation Sciences, University of Virginia Health System, Charlottesville Virginia 22908
4 Heart Foundation Research Centre, Griffith University Gold Coast Campus, Southport, Queensland 4217, Australia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Transgenic mice with cardiac-specific overexpression of adenosine A1 receptors (A1AR) have demonstrated metabolic and functional tolerance to myocardial ischemia. We utilized cDNA microarrays to test the hypothesis that the cardioprotective mechanism(s) of A1 overexpression involves altered gene expression. Total RNA extracted from the left ventricles from A1 transgenic (n = 4) and wild-type (n = 6) mice was hybridized to Affymetrix mgU74A chips. Comparison of RNA expression levels in transgenic to wild-type myocardium revealed ~636 known genes with expression significantly altered by greater than 25%. We observed increased expressions of genes including NADH dehydrogenase, the GLUT4 glucose transporter, Na-K-ATPase, sarcolemmal KATP channels, and Bcl-xl in A1AR-overexpressing hearts. We also observed decreased expression of pro-apoptotic genes including a 50% reduction in message level of caspase-8. Protein expression of GLUT4 and caspase-8 was also altered comparable to the differences in gene expression. These data illustrate genes with chronically altered patterns of expression in A1 transgenic mouse myocardium that may be related to adenosine receptor overexpression-mediated cardioprotection.

microarray; myocardial protection; transgenic animals


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
MYOCARDIAL ISCHEMIC DAMAGE can be attenuated through activation of A1 adenosine receptors (A1AR) in the myocardium by endogenous adenosine or exogenous agonists. Since the degree of protection is limited by the availability of A1ARs, we have developed lines of transgenic mice with 20- to 200-fold overexpression of cardiac A1ARs to optimize the cardioprotective effect (12). Transgenic mouse hearts demonstrate better functional tolerance and diminished cell necrosis, compared with wild-type controls, in response to global ischemia in isolated perfused heart studies (13). Furthermore, hearts with overexpression of A1ARs show improved cardiac energetics during ischemia, compared with wild-type hearts (7). Finally, we have observed that transgenic animals overexpressing A1ARs have reduced infarct size in response to in vivo regional ischemia (25).

The cardioprotection demonstrated in transgenic myocardium is observed in the absence of a prior stimulus, which suggests that transgenic overexpression of A1ARs confers chronic protection. It is likely that such a chronic protective mechanism involves alteration of protein expression patterns. For example, in the late phase of ischemic preconditioning new protein synthesis is required for cardioprotection (16). Furthermore, it has been demonstrated that adenosine-mediated delayed protection is dependent upon de novo protein synthesis (3). These changes in protein translation also result from changes in gene transcription. Recent studies report both increased and decreased gene expression in response to ischemic preconditioning (4, 8, 22). Adenosine has also been demonstrated to regulate expression of multiple proteins through transcriptional control (6, 17, 19). Therefore, we hypothesized that the mechanisms of cardioprotection in transgenic mice overexpressing cardiac A1ARs resulted from altered gene expression. The purpose of this study was to compare gene expression profiles in transgenic myocardium to wild type and identify genes with altered patterns of expression using a murine genome microarray (Affymetrix).


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Tissue extraction and total RNA isolation.
All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and the work was approved by the Institutional Animal Care and Use committee. Transgenic mice overexpressing the rat A1AR cDNA were produced as described previously (12). Briefly, the full-length rat A1 cDNA was subcloned into a construct containing the {alpha}-MHC promoter with a MEF-2 mutation and the human growth hormone polyadenylation signal. The transgenic line used in this study has demonstrated 30-fold overexpression at 20 wk of age (25). Male transgenic (n = 4) and wild-type (n = 6) mice between 20 and 30 wk of age were anesthetized in a halothane chamber and hearts rapidly excised. The ventricles were removed and frozen in liquid nitrogen. Total RNA was isolated by homogenizing whole hearts in TriPure reagent (1 ml/100 mg tissue) and then extracted with chloroform followed by isopropanol precipitation. Contaminants were removed from the resultant RNA by column purification (RNeasy).

RNA labeling and hybridization.
First-strand synthesis was completed by incubation of purified total RNA with oligo-dT primers, followed by incubation with dNTPs for 2 min at 42°C. Reverse transcriptase was added, and the mixture incubated at 42°C for 1 h. This was followed by a 2-h incubation at 16°C with DNA ligase, DNA polymerase, RNase, and dNTPs for second-strand synthesis. Labeled cRNA was synthesized by incubation of 1 µg cDNA with biotin-labeled ribonucleotides and RNA polymerase for 4–5 h at 37°C and then fragmented by heating. A hybridization cocktail including the fragmented, labeled cRNA, control oligonucleotides, control cRNA, and herring sperm DNA in hybridization buffer was washed over the Affymetrix mgU74A probe array for 16 h. Each hybridization was performed four times. A series of washes with increasing stringency was performed to remove all of the hybridization cocktail, and then probe arrays were stained with streptavidin phycoerythrin (SAPE). The fluorescence intensity was then measured using an Affymetrix scanner, and the fluorescence intensity was normalized to the overall intensity for each chip using the Affymetrix microarray suite and dChip software program (http://www.dchip.org).

Statistical analysis.
Average intensity values for each probe set were obtained from the Affymetrix MicroArraySuite (MAS) 4.0 software package. Intensity values were normalized between chips by matching interquartile ranges of different chips to a baseline distribution. This normalization procedure was performed under the assumption that expression patterns of at least 50% of genes on the chip are invariant among different conditions (10). Negative intensity values due to background noise were thresholded at 1.0 to avoid examination of noninformative expression where the mismatched pairs within a probe set were yielding greater binding than the positive matched pairs of the same probe set. This procedure is recommended for the array data generated from MAS 4.0 by the array manufacturer (Affymetrix). Thresholded intensity values were then log-transformed to make a more stable intensity distribution for statistical tests and to interpret up- and downregulated expression changes symmetrically. Global significance testing was performed on each probe set and a P value less than 0.001 was arbitrarily assigned as the level of statistical significance. We report changes in expression as the ratio of raw intensities from transgenic vs. wild-type myocardium. These values are included in the data tables and in brackets within the text.

Real time PCR.
To verify microarray data, real-time PCR quantitation of GLUT4 message levels was performed with TaqMan probes, as described by others (20). Separate extracts of wild-type (n = 6) and A1AR transgenic (n = 4) total RNA were prepared as described. RT-PCR reaction was performed in 96-well plates in reaction buffer containing 3.0 mM MgSO4 (Platinum Quantitative RT-PCR Thermoscript; Invitrogen), 200 nM primers (sense and anti-sense), 100 nM TaqMan probe, and 5–200 ng/well of total RNA. Assays were performed in a Bio-Rad iCycler according to published protocols (20), to determine the PCR cycle at which an increase in fluorescence intensity is first detected (threshold cycle). Linearity of concentration response curves was achieved at 50 ng for A1AR transgenic RNA and 100 ng for wild-type RNA; therefore, these respective RNA concentrations were used to quantitate GLUT4 message levels. All samples were run in triplicate in a single assay run and triplicates were averaged for final RNA quantitation.

Semi-quantitative RT-PCR.
To verify Na-K-ATPase gene expression differences between wild-type and A1AR transgenic myocardium, message for the ATPase and ß-actin were amplified in the same reaction tube and ATPase levels were quantitated as a ratio to ß-actin. Total RNA (1 µg) was reverse transcribed using oligo-dT primers, and 50 ng was amplified using primers specific for the ATPase and ß-actin. (Na-K-ATPase primers: forward 5' -GCCTTACAACGACTCCATCCA-3', and reverse 5' -CGGCATTCTACATTCACCTCC-3', 450 bp; ß-actin primers: forward 5'- GATGGTGGGTATGGGTCAGAAGGA-3', and reverse 5' -GCTCATTGCCGATAGTGATGACCT, 650 bp). The amplification reaction was 40 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. PCR reactions were separated in a 1% agarose gel for densitometry to quantitate band volume (AlphaEase system).

Western immunodetection.
Western analyses were performed on total heart proteins to correlate gene expression to protein expression. Briefly, hearts isolated from freeze-clamped wild-type (n = 9) and transgenic (n = 6) mice were homogenized in 10 vol of homogenization buffer plus protease inhibitors. Fifty micrograms from each protein sample was separated by SDS-PAGE gel and transferred to nitrocellulose membrane. GLUT4 protein was detected using a GLUT4-specific primary antibody, and caspase-8 was detected using caspase-8 anti-mouse monoclonal antibody (Cell Signaling Technology). Protein amounts are expressed as a ratio to the amount of {alpha}-actin, to normalize for protein loading.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Comparison of software analysis packages.
A total of eight hybridizations, four wild-type controls and four transgenic, were completed. The chips were hybridized on two separate occasions, with two wild-type and two transgenic hybridizations performed in each series. Although both series of hybridizations were performed using the Affymetrix mgU74A chips, the first series of hybridizations was performed on chips which contained 3,000 faulty probe sets. These probes represented expressed sequence tags (ESTs) from the IMAGE clone bank and were removed from the analysis. Finally, an adjustment was made to the Affymetrix data acquisition software between the first series and second series of hybridizations, which altered the threshold of probe saturation, thus changing the overall intensity of the chip and therefore the normalization factor of individual chips. These data were therefore analyzed using both the Affymetrix microarray Suite software package and the dChip software package (http://www.dchip.org). The latter software package allows for manual normalization of the data and therefore overrides the PMT adjustment. These two groups of analyzed data were evaluated for consistency by comparison of rank order fold changes in probe intensities and overall distribution of intensity (data not shown). We determined from these comparisons that the data analyzed by both software programs were consistent, and therefore we present here the results of the Affymetrix Microarray Suite software.

Comparison of overall changes in gene expression.
Initial comparison of RNA expression levels in transgenic compared with wild-type myocardium at baseline revealed ~1,285 genes with significant changes in expression. Of the genes with greater than 25% differences in message level, 636 were genes with known identities while 649 were ESTs. For the purposes of this article, data analysis was limited to the identified genes.

A complicating factor in microarray analyses is the frequent occurrence of false positives, defined as single aberrantly high intensity value which increases the overall intensity for a single probe set (15), giving the false impression that the gene is present in the sample. For this reason, the data was manually examined to identify probe sets with three values at or below threshold intensity and one value above threshold intensity. These probe sets were discarded in their entirety. In this study, the rate of false positives detected through manual evaluation of the raw data was 5%. Furthermore, compared with a control sample, a low intensity of the signal will inflate the fold difference in expression. Therefore, any probe set with an overall intensity of less than 100 (an intensity value of 30 is considered a present signal) was also discarded. The remaining 636 probe sets were considered statistically sound and were significantly altered in transgenic myocardium compared with wild-type myocardium. Specific changes in message levels from various protein families are highlighted below.

Expression of mRNAs encoding proteins regulating gene transcription.
This study has examined the effect of transgenic overexpression of cardiac A1ARs on gene expression of multiple transcription factor pathways (Table 1). Message level for Zn-15 zinc transcription factor was increased 7.6-fold in transgenic hearts. Other factors that had increased gene expression levels were {gamma}-interferon (271%) and cAMP response element (174%). An RNA-dependent protein kinase, PKR, which is a mediator of NF-{kappa}B activation, has nearly threefold message levels in transgenic compared with wild-type hearts. Comparison of gene expression patterns in wild-type mouse hearts with transgenic hearts also revealed altered message levels of members of the JAK/STAT pathway and its effectors. Tyk2, a JAK family member, is downregulated by 65% in A1AR transgenic mouse hearts.


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Table 1. Ratio of gene expression in transgenic hearts compared with wild-type controls for genes encoding proteins involved in regulation of transcription

 
Altered expression of genes encoding proteins involved in ATP production and consumption.
These studies have identified proteins involved in cardiac energetics with altered expression in transgenic hearts compared with wild-type controls. Specifically, the GLUT4 glucose transporter (2.40) and NADH dehydrogenase (5.60) were significantly overexpressed in transgenic hearts. These are of particular note because overexpression of these proteins could result in improved cellular ATP production, and we have previously demonstrated preserved ATP levels during an ischemic insult in A1AR transgenic mice (7). Proteins whose activity is dependent upon ATP levels were also increased in A1AR transgenic myocardium. Specifically, these studies identified overexpression of message for sarcolemmal KATP channel subunit (Kir6.2, 1.5-fold) and Na-K-ATPase (2.3-fold). Furthermore, mitochondrial proteins which may also be involved in the myocardial energetic state were overexpressed by 60–130%, included an ATP-binding cassette (ABC7) and voltage-dependent anion channel 1 (VDAC).

Altered gene expression in signal transduction proteins.
Expression analysis of transgenic myocardium identified mediators of several signal transduction cascades (Table 2, Fig. 1) with altered message levels. Of particular note, transgenic hearts had decreased expression of the coding sequences for the Gß subunits (0.22) and adenylyl cyclase (0.47). The downregulation of these mediators is consistent with the demonstrated inhibition of ß-adrenergic activity in A1-overexpressing mice (5). The message levels of other plasma membrane receptors including muscarinic M4 (2.75) and 5-hydroxytryptamine (0.47) receptor were also significantly increased and decreased, respectively. Second messengers involved in the mitogen-activated protein kinase (MAPK) cascade had altered gene expression, including MAPK kinase kinase (MEKK1; 2.65), ERK 1 (1.36), and MAPK interacting kinase (Mnk2, 0.07). These studies also examined the message levels of sulfonylurea receptors (SUR) and potassium channel subunits (Kir), which form the ATP-sensitive potassium channel, another proposed effector of adenosine-mediated cardioprotection. The gene encoding Kir6.2 is upregulated by 61%, whereas there was no difference in the expression of the SUR2 gene. The observed altered message levels of other ion channels and regulators of membrane potential are detailed in Table 3.


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Table 2. Ratio of gene expression in transgenic hearts compared with wild-type controls for proteins involved in signal transduction pathways

 


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Fig. 1. Gene expression differences in proteins involved in intracellular signal transduction. Change in gene expression of representative signal transduction intermediates is expressed as a ratio of expression in transgenic (TG) myocardium to wild-type (WT) myocardium. Positive values indicate genes which are overexpressed in transgenic hearts compared with wild type. Negative values indicate genes which are underexpressed in transgenic hearts compared with wild type. Black bars indicate genes encoding proteins which have been implicated as mediators of cardioprotection.

 

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Table 3. Ratio of gene expression in transgenic hearts compared with wild-type controls for ion channels and regulators of membrane potential

 
Gene expression of cardiac structural proteins and mediators of cardiac remodeling.
Cardiac-specific overexpression of A1ARs resulted in altered expression of genes for 18 myocardial structural proteins (Table 4). We identified significant increases in expression of collagen (type 4), slow myosin heavy chain (ß-subunit), and ß-actin. Gene expressions for troponin I and slow skeletal muscle troponin were downregulated in transgenic hearts (Table 4). We have also identified significant changes in matrix metalloproteinase-4 (8.9-fold overexpressed), a tissue inhibitor of metalloproteinase (TIMP2, 0.32), and endothelin-1 (0.43). These proteins regulate transcription of structural proteins and influence the overall cardiac phenotype and may also control cardiac remodeling and cardiac hypertrophy.


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Table 4. Ratio of gene expression in transgenic hearts compared with wild-type controls for cardiac structural proteins

 
Gene expression changes in the nitric oxide and inflammatory pathways.
Message level of inducible nitric oxide synthase (iNOS), found predominantly in macrophages, was decreased by 50% in A1AR-overexpressing hearts (Table 5). Nitric oxide (NO) is an important intermediate involved in ischemia-reperfusion injury and myocardial protection by ischemic preconditioning and A1AR activation (1). Soluble guanylyl cyclase, a downstream effector of NO, was also underexpressed in transgenic myocardium. Nitric oxide synthases are under the transcriptional regulation of cytokines such as interleukin-1ß (IL-1ß) through the c-Jun and p38 kinase cascades (9). Although no IL-1ß expression was observed in either wild-type or transgenic myocardium, transgenic hearts had overexpression of intermediates of the p38 kinase cascade (MEKK1, 2.65), which may ultimately alter iNOS transcription.


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Table 5. Ratio of gene expression in transgenic hearts compared with wild-type controls for mediators of inflammation

 
The Affymetrix murine GeneChip contains probe sets relating multiple families of immune response elements, which were analyzed to determine the effect of transgenic overexpression of A1ARs on mediators of inflammation (Fig. 2, Table 5). In general, both increases and decreases in gene expression for inflammatory mediators were observed, with a greater preponderance of genes being downregulated in transgenic hearts. These studies have identified altered expression in transgenic myocardium of receptors for IL-7 (2.25), IL-13 (1.57), IL-2 (0.21), IL-10 (0.24), and IL-3 (0.52). Expressions of genes for interleukins IL-12 (2.3), IL-10 (0.42), and IL-2 (0.47) were also significantly altered in transgenic hearts. Macrophage inflammatory protein-1 (MIP-1), an inflammatory mediator, had 57% decrease in gene expression in transgenic hearts compared with wild-type controls.



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Fig. 2. Gene expression differences in proteins involved in initiation and mediation of inflammatory response. Change in gene expression of representative inflammatory mediators is expressed as a ratio of expression in transgenic myocardium to wild-type myocardium. Positive values indicate genes which are overexpressed in transgenic hearts compared with wild type. Negative values indicate genes which are underexpressed in transgenic hearts compared with wild type.

 
Gene expression changes in the programmed cell death pathway.
In these experiments, we compared expression levels of mitochondrial and extramitochondrial proteins involved in apoptosis in wild-type and transgenic mouse hearts (Table 6, Fig. 3). Apoptosis is a major cause of cell death as a result of both acute and chronic ischemia (4a). Apoptosis has been suggested as a significant site of action in cardioprotective mechanisms, and involves a complex set of pro-apoptotic and anti-apoptotic proteins. We observed altered gene expression for both pro-apoptotic and anti-apoptotic proteins. Particular anti-apoptotic proteins upregulated in transgenic mouse hearts included caspase-9S, which was overexpressed by 50% and apoptosis inhibitory protein, which was expressed 2.7-fold greater in A1AR hearts. Also overexpressed in transgenic hearts was CPP32, the enzyme which cleaves preprocaspase-3 to activate caspase-3. Pro-apoptotic proteins with underexpressed genes in A1AR hearts included caspase-8 (0.54), HSP-70 family members (1.48), and caspase-6 (0.41).


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Table 6. Ratio of gene expression in transgenic hearts compared with wild-type controls for proteins involved in programmed cell death pathways

 


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Fig. 3. Gene expression differences in proteins involved in programmed cell death. Change in gene expression of representative apoptotic mediators is expressed as a ratio of expression in transgenic myocardium to wild-type myocardium. Positive values indicate genes which are overexpressed in transgenic hearts compared with wild type. Negative values indicate genes which are underexpressed in transgenic hearts compared with wild type. Black bars indicate genes encoding pro-apoptotic proteins, white bars indicate genes encoding anti-apoptotic proteins.

 
Changes in expression of GLUT4 glucose transporter.
To confirm the data obtained from the microarray studies, GLUT4 gene expression was determined by real-time RT-PCR with RNA isolated from a separate group of hearts. Standard curves were prepared with 5, 25, 50, 100, and 200 ng total RNA from wild-type and transgenic hearts to determine the linear range of the curve (Fig. 4A). Subsequently, GLUT4 message levels were determined in 100 ng RNA from wild-type hearts and 50 ng RNA from transgenic hearts. The average threshold cycle was determined to be 28.99 ± 0.15 in 100 ng wild-type RNA and 29.26 ± 0.09 from 50 ng transgenic RNA, indicating a twofold increase in GLUT4 message levels in transgenic hearts compared with wild-type controls. Western immunodetection revealed GLUT4 expression (as ratio to actin) 1.1 ± 0.2 in wild-type total heart proteins compared with 1.97 ± 0.1 in transgenic myocardium (Fig. 4, B and C). This indicates that the difference in GLUT4 protein expression, 1.83-fold greater in A1AR transgenic myocardium compared with wild-type controls, strongly correlates with differences in gene expression.



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Fig. 4. Overall differences in expression of GLUT4 glucose transporter. Gene expression of the GLUT4 glucose transporter in wild-type and adenosine A1 receptors (A1AR) transgenic myocardium determined by real time RT-PCR (A). The threshold cycle for detection of GLUT4 expression is plotted against the amount of total RNA loaded. Western blot comparing protein expression of GLUT4 in wild-type (n = 9) and A1AR transgenic (n = 6) hearts is shown in B and summarized in C.

 
Protein expression of caspase-8.
Western analysis of caspase-8 in wild-type and A1AR transgenic heart proteins detected three protein bands (Fig. 5): full-length caspase-8 (57 kDa) and cleaved caspase-8 (43 and 18 kDa), which is the active form of the protein. Full-length caspase-8 proteins detected in A1AR transgenic hearts was 0.23 ± 0.02 (ratio to actin) was ~0.70-fold that detected in wild-type hearts (0.33 ± 0.12). Cleaved caspase-8 was similarly decreased in A1AR transgenic hearts compared with wild type (0.18 ± 0.04 for A1AR vs. 0.31 ± 0.10 for wild type). These findings demonstrate that the gene expression differences determined for procaspase-8 result in similar differences in protein expression and eventually lead to comparable decreases in activated protein.



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Fig. 5. Expression changes for caspase-8. Protein expression of caspase-8 was determined in wild-type (white bar) and A1AR transgenic (black bar) hearts with an antibody to total caspase-8, which detected three bands. The 57-kDa band represents the full-length procaspase-8. The 43-kDa and 18-kDa bands are products of caspase-8 cleavage and activation. A shows a representative blot from wild-type (left) and A1AR transgenic (left) myocardium. The ratio of caspase-8 expression to total protein is summarized in B.

 
RT-PCR quantitation of Na-K-ATPase gene expression.
Na-K-ATPase gene expression determined in wild-type hearts was 0.63 ± 0.12 (presented as a ratio to actin) compared with 0.99 ± 0.9 in A1AR transgenic hearts (Fig. 6). These findings confirm that Na-K-ATPase gene expression is slightly increased in A1AR transgenic mouse hearts compared with wild type.



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Fig. 6. RT-PCR detection of Na-K-ATPase RNA expression. A semi-quantitative method of RT-PCR was used to verify Na-K-ATPase gene expression differences between wild-type (left) and A1AR transgenic (right) myocardium. Total RNA reverse transcribed and then amplified with primers specific for the ATPase and ß-actin simultaneously. The message levels of Na-K-ATPase in A1AR transgenic myocardium (normalized to ß-actin message levels) is ~55% greater than that detected in wild-type myocardium.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
The current findings identified about 640 known genes with significantly altered expression levels in hearts with transgenic overexpression of A1ARs, compared with wild-type controls. The genes with altered expressions belong to a variety of protein families including transcription factors, signal transduction pathways, and apoptotic regulatory proteins. The most interesting findings are the increased and decreased expression of genes for the GLUT4 glucose transporter and caspase-8, respectively, which produce similar changes in protein expression. The altered expression of these genes and proteins may contribute to the cardioprotective mechanisms of cardiac-specific A1AR overexpression.

We hypothesized that transgenic cardiac-specific overexpression of A1ARs would likely produce demonstrable changes in gene expression related to cardioprotective mechanisms based upon two lines of evidence: 1) delayed cardioprotection by preconditioning or A1AR activation relies upon de novo protein synthesis (3, 16), which in turn is often dependent upon altered gene transcription; and 2) adenosine receptor activation has been shown to alter gene expression of atrial natriuretic peptide (ANP) (17), Na+-I- symporter (6), MIP-1{alpha} (21), and IL-6 (19). In the current study, we have also demonstrated a decrease in the transcript level for MIP-1{alpha} and an increase in gene expression of preproANP with increased levels of adenosine receptors.

With microarrays, gene expression in innumerable systems and pathways can be examined in a single experimental model. However, this powerful tool does not measure the physiological significance of these findings. Correlating changes in gene expression to other findings, to changes in protein expression and to differences in protein activity, is the best means for establishing the overall physiological significance of an altered gene expression. Several of the current findings can be supported with data from our own previous studies characterizing the A1AR-overexpressing mice. Most notably, we observed a fivefold increase in expression of NADH dehydrogenase and 2.4-fold increase of the GLUT4 transporter gene. We have also confirmed the changes in gene expression using real-time PCR and demonstrated comparable alterations of protein level. GLUT4 is an important transporter for the entry of glucose into myocardial cells, and NADH dehydrogenase is a mitochondrial enzyme involved in the first step of the electron transport chain. The role of these proteins in cellular ATP production supports the hypothesis that their overexpression could contribute to the preservation of ATP stores during ischemia (7) and possibly the overall tolerance to ischemia-reperfusion injury afforded by A1AR overexpression (12, 13, 25). Another previous finding in transgenic mice is the observation of altered adrenergic responsiveness of L-type calcium channel current in myocytes isolated from transgenic hearts (unpublished results). In wild-type cardiomyocytes, A1AR activation diminished calcium current stimulated by isoproterenol. However, A1AR activation enhanced isoproterenol-stimulated calcium currents in transgenic cardiomyocytes. In the current study we have observed a nearly 85% decreased gene expression of L-type calcium channels in transgenic hearts. Although this finding would suggest transgenic myocardium may have fewer calcium channels and therefore decreased calcium current, we also determined a 50% decrease in message level for protein kinase A (PKA), which phosphorylates and inactivates L-type current. Therefore, the decrease in PKA gene expression in transgenic mouse hearts could result in decreased inactivation of L-type calcium currents and therefore increased intracellular calcium available for myocardial contraction.

Altered expression of genes and proteins is a significant mechanism of protection against myocardial ischemic injury. Expression of genes such as glutathione-S-transferase and Bax are altered during myocardial ischemia (11, 22), and myocardial ischemic preconditioning increases complement gene expression. Ischemic preconditioning also increases myocardial protein synthesis, which is necessary for protection against an ischemic insult 24 h later (16). Subsequent studies have identified specific proteins with altered expression associated with a cardioprotective mechanism. Adenosine receptor activation increases expression of Bcl-2 and decreases expression of Bax and results in reduced apoptosis and decreased infarct size following ischemia/reperfusion (27). In the current study, we further demonstrate gene and protein expression changes in GLUT4, Na-K-ATPase, and caspase-8, which may be associated with cardioprotection with A1AR overexpression. GLUT4 is the most abundant glucose transporter and responds to both insulin stimulation and myocardial ischemic stress (26). Several studies have suggested that GLUT4 expression may play an important role in functional and metabolic tolerance to myocardial ischemia and reperfusion injury (14, 23). Furthermore, in mice overexpressing Akt, the functional and necrotic response to ischemia/reperfusion is decreased. Na-K-ATPase is the sarcolemmal membrane pump responsible for maintaining intracellular sodium concentrations and membrane potential, which in turn control cellular volume, intracellular calcium, and myocardial excitation (2). Na-K-ATPase activity is decreased in reperfused myocardium, an effect which may contribute to calcium overload and cellular injury (24). Caspase-8 is an important contributor to cardiomyocyte apoptosis during reperfusion following an ischemic interval (18), and therefore decreased caspase-8 expression and activity could decrease the overall apoptotic loss of cardiomyocytes following ischemic injury. In our hearts with overexpression of A1ARs, we determined a decrease in the expression of the inactive procaspase-8 protein. Western analysis also demonstrated that the cleaved, activated form of caspase-8 is similarly decreased. Although the specific changes in activity of these proteins have yet to be correlated with the cardioprotection with A1AR overexpression, these are the first studies to suggest such an association.

In summary, these studies have identified genes with altered basal expression in transgenic A1AR-overexpressing mouse hearts. The most interesting findings are the overexpression of GLUT4 and the decreased protein expression and activation of caspase-8 which may be involved in the cardioprotective mechanisms of cardiac-specific A1AR overexpression.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. R. Lankford, Dept. of Pediatrics, Univ. of Virginia, Health System, Box 801356, Charlottesville, VA 22908 (E-mail: arl2b{at}virginia.edu).

10.1152/physiolgenomics.00008.2002.


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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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