1 Departments of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
2 Cardiovascular Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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extracellular matrix; integrin; ventricular remodeling; signal transduction; gene expression profiling
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INTRODUCTION |
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The pathophysiological relevance of the FN-induced, integrin-mediated pathway is highlighted by the observation of increased deposition of FN in the extracellular matrix in animal models of cardiac hypertrophy and in patients with cardiac failure (5, 20, 22, 29, 42, 45). The increased synthesis of FN is in part mediated by stimulation of cardiac fibroblasts by angiotensin II, produced in response to mechanical overload (11, 20, 26, 52). The extracellular matrix remodeling associated with interstitial fibrosis can lead to contractile and diastolic dysfunction and consequent heart failure. In contrast to the development of fibrosis in the hypertrophic heart, the gene expression effects of increased FN deposition and consequent integrin activation have been less appreciated. That integrin-mediated pathways are critical for the response to hypertrophic stimuli is suggested by the observations that inhibition of outside-in integrin signaling blocks induction of the hypertrophic phenotype caused by phenylephrine (51, 56) or by mechanical stretch (1, 33).
In this study, we examined the changes in gene expression induced by FN outside-in signaling in NRVM using high-density oligonucleotide microarrays. The results obtained provide a framework to analyze in a comprehensive manner the transcriptional response to integrin activation and suggest novel pathways activated by FN in hypertrophic cardiac myocytes.
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MATERIALS AND METHODS |
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Immunofluorescence and image analysis.
Cardiac myocytes were cultured in 8-well glass chamber slides (Lab-Tek II; Nalge Nunc, Naperville, IL) coated with gelatin or FN, as described above. After 4 days of culture, the cells were washed with phosphate-buffered saline (PBS), fixed in PBS containing 2% paraformaldehyde for 20 min, and permeabilized with pH 7.4 PBS containing 0.1% Triton X-100 for 20 min. Nonspecific binding was blocked by incubation for 1 h at room temperature with PBS containing 0.1% Tween 20 (PBS-T) and 5% normal donkey serum. The following primary antibodies were diluted in PBS-T with 5% goat serum and incubated for 1 h at room temperature: 1) rabbit anti-ANP (Peninsula Laboratories, Belmont, CA) and 2) mouse monoclonal anti-sarcomeric -actinin (Sigma, St. Louis, MO). The slides were then briefly rinsed in PBS-T and washed three times for 5 min in PBS-T. Cy2-, Cy3-, or Cy5-conjugated AffiniPure donkey secondary antibodies (anti-mouse or anti-rabbit), affinity purified to remove cross-reactivity with IgG from other species, were from Jackson ImmunoResearch Laboratories (West Grove, PA). The secondary antibodies were used at 1/300 dilution in PBS-T with 5% donkey serum. In selected samples, rhodamine-phalloidin (Sigma, St. Louis, MO) was also added at 100 ng/ml for visualization of the actin cytoskeleton and sarcomere organization. After a 45-min to 1-h incubation at room temperature, the slides were washed three times for 5 min with PBS-T and were mounted in 50% glycerol in PBS containing 1 µg/ml bis-benzimide (Hoechst 33258) nuclear stain. The cells were visualized in a Zeiss Axioplan 2 confocal imaging system or in an Olympus BX40 epifluorescence microscope. Images were acquired with a SPOT camera, and image analysis was performed with Photoshop (Adobe Systems, San Jose, CA). To validate double or triple labeling experiments, appropriate negative controls omitting one of the secondary or primary antibodies were performed to rule out unwanted cross-reactivity between the various primary and secondary antibodies and to rule out optical leaching between the different filter combinations. For cell surface area measurements, several pictures were taken from representative fields using Nomarski interference optics on culture dishes or
-actinin immunofluorescence on fixed cells. At least 200 myocytes for each data point were analyzed with the NIH IMAGE software.
RNA purification and real-time RT-PCR.
Cells were cultured as described above, and RNA was purified using TRIzol (Invitrogen, Carlsbad, CA), according to manufacturer instructions. RNA was precipitated from the TRIzol reagent with isopropanol and resuspended at 0.51.0 mg/ml using RNASecure solution (Ambion, Austin, TX). RNA was treated with DNase I (DNA-free, Ambion) before reverse transcription. For reverse transcription, 60 ng of RNA were mixed with 5 µM random hexamers, 1 mM each dNTP, 7.5 mM MgCl2, 40 U RNasin (Promega, Madison, WI), 1x PCR buffer II (Applied Biosystems, Foster City, CA) and 250 U of SuperScript II reverse transcriptase (Invitrogen). The reaction was incubated at 25°C for 10 min, 48°C for 45 min, and 95°C for 5 min, then cooled to 4°C. For SYBR Green quantitative real-time PCR, 2 µl of reverse transcription reaction was mixed with 400 nM each specific primer (see Table 2) and 1x SYBR PCR Master Mix (Applied Biosystems). The reaction was incubated in a model 7100 thermocycler (Applied Biosystems) for 45 cycles consisting of denaturation at 95°C for 15 s and annealing/extension at 5860°C for 1 min. The critical cycle (CT) was determined by the ABI Prism 7000 SDS software, and quantification of relative mRNA levels was performed by the software using a calibration curve obtained by serial dilutions of a standard RNA preparation from rat heart.
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Data analysis.
GeneChip expression software (Affymetrix, Santa Clara, CA) was used to determine the absolute analysis metrics using the probe arrays hybridization measure after scanning the arrays, as directed by the Affymetrix GeneChip standard procedure. The average difference for each probe set is an average of the differences between the "perfect match" (PM) and control "mismatch" (MM) probe intensities and is directly related to the level of expression of the transcript. The GeneChip Detection Algorithm was used to calculate a detection P value using the distance between the discrimination score [R = (PM MM)/(PM + MM)] and the threshold Tau = 0.015, tested by a one-sided Wilcoxon signed rank test. Only those probe sets with detection P values <0.05 were called "present" and considered for further analysis in this study. The level of expression of each transcript (signal) was determined by GeneChip using a one-step Tukey biweight estimate. The data points for each gene of the FN or control arrays were normalized using a targeted normalization procedure in which the average signal from the 3' GAPDH probe sets was defined as unchanged. The signals from all the experimental arrays were then multiplied by a normalization factor to obtain similar average intensity between arrays for the 3' GAPDH probe sets. This normalization method was chosen because the global increase in gene expression associated with hypertrophy precludes global or even selected "housekeeping" gene normalization procedures. We selected GAPDH because it has been broadly used as an invariant control for mRNA expression in studies of cardiac myocyte hypertrophy, and in our experience GAPDH mRNA expression by Northern blot or real-time RT-PCR shows very little variation in cells plated in the presence or absence of FN and other hypertrophic agents (unpublished observations). The corrected signals after the normalization procedure were then used to determine the fold change of FN relative to control by dividing the signal of each probe set in the FN array by respective signal in the control array. Each experiment was performed in duplicate, and the fold change ratios from each replicate were averaged in the final analysis. The complete data set was deposited into the NCBI Gene Expression Omnibus (GEO) database with the accession number GSE1055.
Pathway analysis.
Pathway analysis was performed using the GenMAPP software version 1.0 (Gladstone Institutes, UCSF, San Francisco, CA) (12), downloadable from http://www.genmapp.org. This software uses an identifier for each probe set to display the expression level of each gene in a pathway. We used the Swiss-Prot identifiers for each gene based on the annotation data provided by Affymetrix, as well as a table relating rat GenBank identifiers to Swiss-Prot numbers obtained with the Dragon program (http://pevsnerlab.kennedykrieger.org/annotate.htm). When Swiss-Prot identifiers were not available, the GenBank identifier was used and replaced in the respective gene slot in the pathway. The annotation for each probe set of the RAE230A array was verified by downloading the latest annotation files from Affymetrix. In addition, we performed BLAST analysis using the probe target sequence described by Affymetrix for all the probe sets with significant levels of expression (present) where there were discrepancies between various probe sets corresponding to the same transcript or where the annotation was "transcribed sequences." When genes in a pathway were not automatically found in the array with the GenMAPP software, we used NCBI BLASTN (http://www.ncbi.nlm.nih.gov/BLAST/) and BLAT alignments to the UCSC rat genome browser (http://genome.ucsc.edu)(27) to identify sequences homologous to the probes in the array. Using these strategies, we assigned a specific gene (with the corresponding HUGO Gene Nomenclature Committee approved gene symbol) to 7,250 RAE230A probe sets corresponding to 5,664 different genes, compared with the original 5,193 probe sets corresponding to 4,662 different genes from the Affymetrix annotation files. Moreover, in several instances, the original annotation was corrected based on the results of BLAST analysis.
All the pathways in the GenMAPP database were analyzed to determine whether a significant number of genes in each pathway were affected by FN vs. control (15). We examined the frequency of genes fulfilling a certain criterion (such as increase 1.5) in each group or pathway and compared with null hypothesis expected frequency for that group or pathway based on the total number of genes examined on the array. The z-score was derived by dividing the difference between the observed number of genes meeting the criterion in a specific group or pathway and the expected number of genes based on the total number of genes in the array meeting the criterion and standardizing by dividing by the standard deviation of the observed number of genes under the hypergeometric distribution. The equation used was:
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A positive z-score indicates that more genes than expected fulfilled the criterion in a certain group or pathway; therefore, that group or pathway is likely to be affected by FN.
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RESULTS |
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Among the cytoskeleton-associated genes, tubulins and tubulin-associated genes were significantly upregulated (z = 3.9), whereas actin-related genes as a group were not (z = 1.2). However, individual genes, such as skeletal--actin (Acta1), were significantly induced. Myosins and tropomyosins are a heterogeneous group with highly expressed sarcomeric-specific genes and a large variety of nonsarcomeric myosins. Among the highly expressed myosins and tropomyosins (normalized levels >200), a significantly larger proportion of genes was upregulated (z = 2.8). These include tropomyosins 1, 2, and 3 (Tpm1-3), ßMHC (Myh7), cardiac myosin-binding protein C (Mybpc3), cardiac myosin light chains (Myl2, Myl3, Myl4), and smooth-muscle myosin light chain (Myl6).
Three metabolic pathways achieved high z-scores: cholesterol biosynthesis (z = 8.9), fatty acid biosynthesis (z = 4.2), and the mitochondrial respiratory chain (z = 6.4). Again, the z-scores are specific for these pathways, and they are much reduced when the groups are widened. For example, considering all genes involved in fatty acid metabolism reduces the z-score from 4.2 to 2.5, suggesting that our statistical analysis correctly identifies significantly coregulated genes specific to the anabolic pathway.
Comparison with an aortic banding mouse model.
Using the LocusLink gene symbol field as a link between the rat and mouse annotation databases and the Resourcerer program for matching Affymetrix GeneChips (http://pga.tigr.org/tigr-scripts/magic/r1.pl), we compared our results to those of aortic-banded mice, published by the Harvard Cardiovascular Genomics group (8, 19) (Table 4 and Supplemental Table S1, available at the Physiological Genomics web site).1
In this mouse model of cardiac hypertrophy, gene expression arrays were done on RNA obtained from hearts at 1 h, 4 h, 24 h, 48 h, 1 wk, and 8 wk postaortic banding and compared with sham-treated control animal (8, 19). We were able to match 4,304 probe sets from the rat RAE230A with 4,440 probe sets in the mouse MG-U74Av2 array, corresponding to 3,316 unique genes, of which 1,727 were expressed by NRVM and 452 were upregulated 1.5-fold in our experiments. Table 4 shows genes upregulated in vivo that were not significantly changed by FN in our purified NRVM. Interestingly, these include genes coding for several extracellular matrix and transmembrane proteins (biglycan, collagens, FN, epithelial membrane protein 1, fibrillin 1, TIMP1) as well as genes possibly associated with noncardiac cell lineages such as B-cell translocation gene 2, macrophage migration inhibitory factor, epithelial membrane antigen, and MARCKS-like protein. These observations highlight one advantage of purified cell culture studies for cell-autonomous pathway identification. Many of the genes identified as upregulated in our studies did not show a change in vivo. One possibility for the discrepancy, in addition to hypertrophic stimulus- and species-specific pathways, is that relatively small changes (<3-fold) in mRNA abundance exclusive to cardiac myocytes may be undetectable when whole heart RNA is examined, since cardiac myocytes may comprise less than 50% of the cells in the diseased heart. Our ability to purify NRVM and detect statistically significant changes as small as 1.5-fold may uncover important pathways missed with whole organ analyses.
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Protein synthesis.
Ribosomal proteins represented the group affected the most by FN, in part because of their large number, resulting in the highest z-score (Table 3). Since many of the ribosomal proteins were also induced in the mouse aortic banding model (Supplemental Table S1), these data suggest that ribosomal protein abundance increases in hypertrophic cardiac myocytes. In addition, we measured significant increases in the mRNA abundance of subunits of translation factors eIF2, eIF3, and eEF1 and in aminoacyl-tRNA synthetases Vars2, Cars, and Sars (Supplemental Table S1). To our knowledge, this is the first report suggesting the involvement of these translation factors in cardiac hypertrophy. Interestingly, elongation factor eEF2 is regulated posttranscriptionally by phosphorylation (7) and showed unchanged mRNA levels in our experiments.
Protein degradation.
We have identified the proteolytic ubiquitin-proteasome pathway as a target for upregulation by FN (Table 3). The z-scores for ubiquitin and proteasome-related genes were 3.4 and 2.6, respectively. These relatively low z-scores reflect the fact that several ubiquitin and proteasome-related genes were not upregulated, or only slightly upregulated, by FN. Two of those genes (Uchl1, Usp18) were also upregulated in vivo (Supplemental Table S1). Uchl1 codes for a carboxy-terminal ubiquitin esterase involved in removing ubiquitin from small adducts, whereas Usp18 is a member of the ubiquitin-specific proteases that disassemble polyubiquitin chains into free monomeric ubiquitin. Additional genes induced by FN and involved in the ubiquitin-proteasome pathway included ubiquitin proteases (Usp1, Usp2, Usp4, Usp10, Usp15, Usp39, Usp49), ubiquitin precursors (FAU, Uba52, Ubb, Ubc), members of the E2-ligase complex (Ube2d2, Ube2j1, Ube2v1, Ube2v2), E3-ligase and cofactors (Sugt1, Rbx1, Rnf7, Spop, Fbxl6, Fbxo7, Fbxo22, Fbxw5, Tceb2, Cop1), and components of proteasome 19S (Psmc2, Psmc3, Psmc5, Psmd1, Psmd9-11, Psmd13) and 20S (Psma7, Psmb3-6, Psmb8, Psmb10) subunits (results not shown).
Metabolic pathways.
Virtually all of the expressed cholesterol biosynthesis genes present in the RAE230A array were significantly induced by FN (Fig. 3). A rate-limiting enzyme, the P-450 cytochrome CYP51, responsible for the sterol 14- demethylase reaction, is coded by one of the genes with the highest level of induction by FN (Table 1). Several genes in the cholesterol biosynthesis pathway (Cyp51a1, Dhcr7, Hmgcs1, and Sqle) were also induced by aortic banding (Supplemental Table S1). We confirmed the induction of several of the cholesterol biosynthesis genes by RT-PCR (Table 2).
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Induction of cholesterol and fatty acid biosynthesis in other cell types has been linked to activation of the transcription factors SREBP1 and SREBP2. Microarray analysis of mouse liver mRNA expression in SREBP1a and SREBP2 transgenic mice and SREBP cleavage-activating protein (SCAP) knockout mice revealed 33 genes that are targets of both SREBP1a and SREBP2, 18 genes activated by SREBP1a but not SREBP2, and 10 genes that are targets of SREBP2 only (23). When we applied the pathway analysis to the 38 SREBP1a target genes represented in the RAE230A array and expressed in NRVM, 32 were induced 1.5-fold by FN (Table 3). These genes include all of the 17 cholesterol biosynthesis pathway genes in Fig. 3, plus the LDL receptor, involved in cholesterol uptake, INSIG1, a repressor of SREBP induced by negative feedback when adequate sterol levels are present in the cell (23), SREBP1 itself, 9 fatty-acid synthesis genes (Me1, Acacb, Acac, Fasn, Acat2, Elovl6, Facl5, Fads1, Fads2), and 3 other genes (Gstt3, Mac30, Tkt).
Respiratory chain.
Induction by FN of several electron transporters and other genes involved in the mitochondrial respiratory chain was observed in our experiments (z = 6.4, Table 3). These include genes in the NADH-ubiquinone oxidoreductase complex I (15 of 25 expressed genes were induced), ubiquinol cytochrome c reductase complex III (7 of 11 induced), cytochrome c oxidase complex IV (10 of 16 induced), and ATP synthase complex V (6 of 16 induced). Complex IV includes Cox17, a protein essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion, which was induced 1.9-fold by FN. Interestingly, other genes involved in regulation in copper transport and metabolism were also induced by FN, including Slc31a1, a copper transporter, and Atox1, a copper chaperone (Supplemental Table S1).
Cytoskeletal and sarcomeric proteins.
Our statistical analysis of pathways affected by FN in cardiac myocytes uncovered significant changes in genes involved in microtubule assembly and sarcomeric proteins (Table 3). FN induced -tubulin genes Tuba1 and Tuba4, ß-tubulin genes Tubb, Tubb2, and Tubb5, as well as microtubule-associated proteins Mtap1a and Mtap4 (Supplemental Table S1) and tubulin- and microtubule-maintenance chaperones, including T-complex subunits 1, 3, 4, 5, and 7 and tubulin cofactors A, B, D, and E (not shown).
In contrast to microtubule proteins, actins and actin polymerization genes as a group were not significantly altered by FN (z = 1.2). However, several genes playing important roles in actin polymerization were upregulated, including Actn1, Cfl1, Cnn2, Coro1c, Cyr61, Enah, Fbln2, and Tagln, drebrin 1, thymosin b4x, filamin C, and Arp2/3 complex members (Supplemental Table S1). Interestingly, melusin, a ß1-integrin binding protein linking mechanical stretch and cytoskeletal dynamics in cardiac myocytes (6), was induced 2.1-fold by FN. These results suggest that changes in actin polymerization and cytoskeleton dynamics may result from FN-induced cardiac hypertrophy.
Genes coding for sarcomeric proteins were also significantly induced by FN (Table 3 and Supplemental Table S1). These include the fetal isoforms SkA (Acta1) and cardiac ßMHC (Myh7), well known to be upregulated in cardiac hypertrophy, as well as myosin light chains, cardiac myosin-binding protein C, troponin C and T, and tropomyosin genes (Supplemental Table S1). The tropomyosin genes are regulated by alternative splicing in a tissue-specific manner (57), and the RAE230A array probe sets can distinguish some of the alternatively spliced isoforms. For example, probe set 1370287_a_at recognizes the smooth muscle-specific exon 9d and showed an expression level of 1,157 and induction of 1.3-fold by FN, whereas probe set 1368724_a_at recognizes the striated muscle-specific exon 9b and showed an expression level of 16,414 and induction of 2.1-fold by FN in cardiac myocytes. Our results suggest that transcriptional upregulation of these sarcomeric genes beyond the general increase in transcription accompanying hypertrophy underlie not only increased sarcomeric assembly, but also actual shifts in the isoform composition of the sarcomeres.
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DISCUSSION |
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One important feature of hypertrophic cardiac myocytes is increased protein synthesis, achieved in part by an increase in translational capacity. The increase in translational capacity is supported by our observation of FN-induced expression of ribosomal protein genes, resulting in the highest z-score of the pathways examined (Table 3). It is known that hypertrophy and integrin activation increases the translational efficiency of ribosomal protein mRNAs (3, 54). Therefore, it is likely that the abundance of ribosomal proteins increases during hypertrophy due to increased transcription and increased translation. Together with increased transcription of the 45S rRNA precursor (2, 36), the resulting increased translational capacity allows for enhanced protein synthesis required for hypertrophic growth.
In addition to the increase in translational capacity, hypertrophic cardiac myocytes are characterized by increased translational efficiency. The rate-limiting step of regulation of translation efficiency involves the regulation of the activity of translation initiation factor eIF4E, which modulates the formation of the eIF4F complex and affects the affinity of mRNA for the ribosome. We did not detect a change in mRNA abundance of eIF4E, but this protein is mostly regulated by phosphorylation and association with 4E-binding proteins in cardiac myocytes (55). However, we did measure increases in mRNA levels of eIF2 and eIF3, which are involved in recruitment of capped mRNAs to the ribosome, eEF1 subunits 1, ß,
, and
, which bind aminoacyl-tRNA complexes (7), and valine, serine, and cysteine-tRNA synthetases, suggesting that synthesis of certain aminoacyl-tRNA and their recruitment to the ribosome are limiting in hypertrophic cardiac myocytes.
The robust increase in protein synthesis during cardiac myocyte hypertrophy is accompanied by moderate increases in protein degradation, resulting in higher protein turnover and net accumulation of proteins. The ubiquitin-proteasome pathway plays the major role in proteolytic degradation of misfolded or damaged proteins, turnover of short-lived proteins, and also of sarcomeric proteins (17). We have identified the ubiquitin-proteasome pathway as a target for upregulation by FN (Table 3), suggesting that the ubiquitin-proteasome pathway plays a role in protein turnover in hypertrophic cardiac myocytes and that inhibition of this pathway may modify the hypertrophic phenotype.
Consistent with the growth in cell volume, the hypertrophic cardiac myocytes require increased membrane assembly, which is reflected by a coordinated induction in cholesterol biosynthesis. Virtually all of the cholesterol biosynthesis genes detected in the RAE230A array were significantly induced by FN (Fig. 3). This is a novel observation in cardiac myocytes and may be a mechanism by which cholesterol synthesis inhibitors prevent cardiac hypertrophy (41, 44), in addition to their effect in the isoprenylation of small G proteins, such as Ras and RhoA (30).
Although the downregulation of fatty acid oxidation in cardiac hypertrophy has been extensively reported, the ability of cardiac myocytes to synthesize and elongate fatty acids has been poorly studied, since the heart is thought incapable of significant fatty acid synthesis (16, 21). However, the ratio of C18 to C16 fatty acids in the heart is twice that of plasma (10), purified cardiac myocytes were able to desaturate and elongate labeled linoleic acid (34), and there is no satisfactory explanation for the fate of considerable amounts of malonyl-CoA in the heart (21). In our NRVM cultures, we have detected significant mRNA expression and induction by FN of fatty acid synthetase (Fasn) and other genes in the fatty acid biosynthesis pathway. Other studies have detected Fasn mRNA in the heart and reported regulation by starvation (28), glucocorticoids (48), and thyroid hormone (4). It is possible that in the perfused adult heart (16, 21) fatty acid synthesis is undetectable, but in cultured NRVM fatty acid synthesis plays a role in hypertrophic growth.
Since all of the cholesterol biosynthesis genes and nine fatty acid biosynthesis genes induced by FN are also known targets of transcription factor SREBP1 (23), our data suggest that SREBP1 may be co-coordinating the induction of these two pathways to maintain the proper balance of cholesterol and phospholipid-associated fatty acids in hypertrophic cardiac myocytes. We are currently testing the hypothesis that activation of SREBP1 during cardiac hypertrophy is responsible for the induction of transcription of cholesterol and fatty acid biosynthesis genes.
Induction of respiratory chain components has not been well studied in hypertrophic myocytes. ATP synthase activity is induced by increased contraction and inotropic agents in cardiac myocytes (13), and ATP synthase subunit c mRNA was induced by norepinephrine in the rat heart (32). In our experiments we observed induction of components I, III, IV, and V of the mitochondrial respiratory chain. The highest induction in the ATP synthase complex F1 was observed in subunit e (2.4-fold). Interestingly, regulation of subunit e by hypoxia at the pretranslational level has been previously observed in cardiac myocytes (31). It is possible that transcriptional induction of several limiting components of the respiratory chain is required to meet the increased energy demand of the hypertrophic myocytes.
Given the recently identified role of calcineurin in hypertrophy (37), it is interesting that FN did not induce the expression of any of the isoforms of calcineurin or the calcineurin/NFAT-target gene Dscr1, which can be induced by mechanical stretch or adrenergic stimulation (49, 53). On the other hand, FN induced calmodulin 1 (Supplemental Table S1), which activates calcineurin in response to elevated intracellular Ca2+ levels. FN also induced the calcineurin binding protein myozenin 2 (calsarcin-1), which tethers calcineurin to the Z-line of sarcomeres (18), calcium/calmodulin-dependent protein kinase I, which synergizes with calcineurin in promoting hypertrophy (43), and frequenin homolog, which can substitute for calmodulin and regulate cardiac Ito K+ channels (38). It is possible that FN induces a permissive state for calcineurin activation but is insufficient for the full induction of calcineurin, which may require increased intracellular Ca2+ levels induced by mechanical stretch or adrenergic agonists.
We have identified tubulins and microtubule-associated proteins, such as Mtap4, as targets of FN-induced upregulation. Microtubule density is increased in cardiac hypertrophy and may interfere with contractility and play a role in heart failure progression (25, 46, 47, 50). Mtap4 is a microtubule-stabilizing protein and is thought to be involved in microtubule densification during cardiac myocyte hypertrophy (46, 50). Interestingly, we also observed induction by FN of several chaperones implicated in tubulin and microtubule maintenance. These results suggest that both pretranslational and posttranslational mechanisms are involved in tubulin upregulation and microtubule stabilization in hypertrophic cardiac myocytes.
FN induced the connective tissue growth factor (CTGF) gene, coding for a cysteine-rich, glycosylated protein that regulates proliferation of fibroblast and other mesenchymal cells and induces secretion of extracellular matrix proteins. CTGF is induced by TGF-ß in cardiac fibroblasts and myocytes and is thought to play a role in heart fibrosis (9). FN also upregulated WNT1 inducible signaling pathway protein 2 (WISP2), another member of the CTGF family, but its expression in the heart has not been previously reported. Another extracellular protein induced by FN, the secreted acidic cysteine-rich glycoprotein (osteonectin, SPARC), is abundant in remodeling tissues and in diseases associated with fibrosis. It was reported to be induced by ß-adrenergic stimulation in the myocardium of adult rats (35) and in a transgenic mouse model of hypertrophy (24). It is tempting to speculate that secretion of the cysteine-rich proteins SPARC, CTGF, and WISP2 by cardiac myocytes may be responsible in part for the fibroblast proliferation and fibrosis associated with pathological hypertrophy and heart failure in vivo and may explain the increased mRNA levels of various collagen genes, FN, biglycan, and other extracellular matrix genes, which were not induced by FN in purified NRVM but were upregulated in the mouse aortic banding model (Table 4).
In summary, our model of FN-induced hypertrophy together with our statistical analysis of gene expression patterns and comparison with the mouse aortic banding model revealed several pathway and gene expression changes not previously associated with cardiac hypertrophy. Our studies also appear to distinguish cardiac myocyte-specific from non-myocyte-dependent pathways. Further work is required to determine how critical these gene expression changes are for a complete cardiac hypertrophy phenotype and to evaluate how modulation of these changes may affect progression to heart failure.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of H. Chen: Department of Pathology, New York University, New York, NY 10016.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. L. Sepulveda, Dept. of Pathology, Univ. of Pittsburgh, CHP MT 5726, 200 Lothrop, Pittsburgh, PA 15213 (E-mail: sepulvedajl{at}upmc.edu).
10.1152/physiolgenomics.00104.2004.
1 The Supplementary Material for this article (Supplemental Table S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00104.2004/DC1.
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REFERENCES |
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