Published online before print December 24, 2007, doi: 10.1161/HYPERTENSIONAHA.107.098046
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(Hypertension. 2008;51:309.)
© 2008 American Heart Association, Inc.
Original Articles |
Gene Expression Pattern in Biomechanically Stretched Cardiomyocytes
Evidence for a Stretch-Specific Gene Program
From the Department of Internal Medicine III (D.F., C.K., C.H., M.L., H.A.K., N.F.), University of Heidelberg, and the Division of Intelligent Bioinformatics Systems (B.B.), DKFZ, Heidelberg, Germany.
Correspondence to Norbert Frey, MD, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany. E-mail norbert.frey{at}med.uni-heidelberg.de
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Abstract |
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Biomechanical stress ie, attributable to pressure overload, leads to cardiac hypertrophy and may ultimately cause heart failure. Yet, it is still unclear how mechanical stress is sensed and transduced on the molecular level. To systematically elucidate the underlying signal transduction pathways, we analyzed the gene expression profile of stretched cardiomyocytes on a genome-wide scale in comparison with other inducers of hypertrophy such as pharmacological stimulation. Neonatal rat ventricular cardiomyocytes were either stretched biaxially or stimulated with phenylephrine (PE), both resulting in a similar degree of hypertrophy. Microarray analyses revealed 164 genes >2.0-fold up- and 21 genes <0.5-fold downregulated (P<0.01). Differential expression was confirmed by real-time polymerase chain reaction. Genes of the "fetal gene program" such as BNP were induced by both stretch (4.2x) and PE (2.9x). We also verified upregulation of known stretch-responsive genes, including HSP70 (20.9x) and c-myc (3.0x). Moreover, several genes were found to be preferentially induced by stretch, such as the cardioprotective cytokine GDF15 (24.8x) and heme oxygenase 1 (Hmox1, 10.8x; both confirmed on protein level). Neither PE nor endothelin-1 upregulated GDF15 and Hmox1, whereas angiotensin II significantly induced both genes. Conversely, the AT1 receptor blocker irbesartan markedly blunted stretch-mediated GDF15 and Hmox1 upregulation, suggesting that the angiotensin receptor tranduces the biomechanical induction of these genes. In conclusion, we report a comprehensive gene expression profile of cardiomyocytes subjected to biomechanical stress in comparison with pharmacologically induced hypertrophy. Our data imply that a stretch-specific gene program exists, which is mediated, at least in part, by angiotensin II–dependent signaling.
Key Words: hypertrophy • gene expression • microarray analysis • stress • mechanical
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cardiac hypertrophy can be induced either by intrinsic stimuli, ie, by inherited mutations of contractile proteins leading to hypertrophic cardiomyopathy or by extrinsic stimuli such as chronic biomechanical stress attributable to arterial hypertension or valvular heart disease.1 Long standing left ventricular hypertrophy leads to progressive remodeling with fibrosis and cardiomyocyte apoptosis,2 ultimately resulting in ventricular dilation, heart failure, and increased susceptibility to malignant arrhythmias.3 Accordingly, cardiac hypertrophy has been shown to be an independent and powerful predictor of cardiovascular morbidity and mortality.4–6
The typical cellular features of cardiac hypertrophy comprise an increase in cardiomyocyte size associated with enhanced sarcomerogenesis and protein synthesis, as well as activation of the "fetal" or "hypertrophic" gene program, including the natriuretic peptides ANF and BNP.7 However, it is still unclear how biomechanical stress, ie, attributable to pressure overload, is sensed and transduced toward initiation of cardiomyocyte hypertrophy. Recently, it has been proposed that a putative stretch receptor is localized at the sarcomeric Z-disc,8 involving proteins such as muscle LIM protein (MLP)9 and Calsarcin-1.10 Similarly, the integrin-binding proteins Melusin11 and focal adhesion kinase (FAK),12 2 proteins of the adjacent costamere, have been implicated in mechanotransduction. Furthermore, the Na+/H+ exchanger13 and gadolinium-blockable stretch-dependent channels14,15 also mediate mechanically induced hypertrophy. Thus, biomechanical load appears to be a critical stimulus in the induction of myocardial hypertrophy.16,17 However, neurohumoral pathways including adrenergic signals,18 the renin-angiotensin-aldosterone system (RAAS),19 and endothelin-1 (ET1)–dependent signaling20 may also contribute to the development of cardiomyocyte hypertrophy. In an in vivo setting it is therefore often impossible to distinguish between the direct effects of increased biomechanical load and secondary neurohumoral activation. To directly assess the molecular pathways which are involved in stretch-dependent signaling, we set up an in vitro stretch experiment using neonatal rat cardiomyocytes. For comparison, we included cardiomyocytes stimulated by phenylephrine to detect genes with stretch-specific regulation. Using a genome wide gene expression screen, a distinct stretch-induced gene expression pattern could be identified. The differentially regulated genes included several established members of the hypertrophic gene program. Interestingly, we also discovered genes with stretch-specific induction, which could not be induced on phenylephrine or endothelin-1 treatment. Of note, angiotensin II (Ang II) was capable of stimulating these genes, whereas the angiotensin II type 1(AT1) receptor blocker irbesartan blocked stretch-mediated induction. Thus, we conclude that a stretch-specific gene expression program exists, which is mediated, at least in part, via AT1 receptor–dependent signaling.
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Materials and Methods |
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The experimental procedures of the preparation of neonatal cardiomyocytes, cardiomyocyte stretching, RNA isolation and purification, microarray hybridization and microarray data analyses, real-time PCR analyses of gene expression, Northern and immunoblotting, as well as immunofluorescence experiments are provided in the online methods supplement. All results are shown as the mean±SD. Real time PCR data analyses were carried out using the
ct method.21 Statistical significance was determined using ANOVA followed by Bonferroni post-hoc test, 2-sided Student unpaired t test or Fisher exact test. P<0.05 or less was considered statistically significant. |
Results |
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Biomechanical Stress Results in Significant Cardiomyocyte Hypertrophy
Biomechanical stress is a known inductor of cardiac hypertrophy. However, the coactivation of several neurohumoral signaling pathways makes it difficult to delineate a stretch-specific gene expression profile in vivo. To specifically dissect biomechanical stress pathways, we biaxially stretched purified NRVCMs up to 112% of baseline length for 24 hours. Cardiomyocytes treated with 50 µmol/L Phenylephrine (PE) and unstretched cardiomyocytes served as controls. Stretching and treatment with PE resulted in a comparable degree of cellular hypertrophy as shown in immunofluorescence experiments (Figure 1A). In both cases, cell size, sarcomeric organization, as well as expression of the atrial natriuretic factor (Figure 1B) were significantly increased in comparison to unstretched cells. In stretched cells, cell surface area (Figure 1C) was augmented 1.88-fold, P versus control <0.001. Gain in cell surface area in PE-treated cell was very similar (1.85-fold, P versus control <0.001, P versus stretch n.s.). Members of the hypertrophic gene program, such as atrial natriuretic factor (Figure 1B and 1D) and beta myosin heavy chain were also induced to a similar degree both by stretch and phenylephrine (Figure 1D).
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P<0.001 vs control) and stretch-stimulated cells 1.88-fold (
Distinct Gene Expression Profile in Cardiomyocyte Hypertrophy Attributable to Biomechanical Stress
To gain a comprehensive view of differentially regulated genes in response to both biomechanical stress and pharmacological induction of hypertrophy, we performed genome wide gene expression screens using Affymetrix 230.2 rat microarrays. Bayes analyses of the raw data revealed 185 differentially regulated genes in the stretch group (a posteriori probability of >99%). 164 of these genes were significantly and more than 2.0-fold upregulated (Figure 2A and Table S2, available online at http://hyper.ahajournals.org), whereas 21 were significantly downregulated (less than 0.5-fold; Figure 2A and Table S2). In contrast, stimulation with PE resulted in 450 significantly regulated genes (238 genes more than 2.2-fold upregulated and 211 less than 0.45-fold downregulated; Figure 2B and Table S3).
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Next, the resulting data were normalized and displayed in heat maps, revealing highly reproducible results within groups and a low variance between microarray hybridizations (Figure 2C for stretch, Figure 2D for phenylephrine). For genes differentially regulated by biaxial stretch (Figure 2A), hierarchical clustering of the results showed that approximately 50% of these genes were upregulated to a similar degree by PE (upper part of figure 2C). Moreover, a cluster of multiple genes displayed stretch-specific upregulation (Figure 2C, lower half). In contrast, genes downregulated by biomechanical stress were also downregulated by phenylephrine (lowest lanes in Figure 2C). When applying hierarchical clustering to genes significantly regulated by phenylephrine (Figure 2D), we again observed that the gene expression profiles of stretch- and PE-induced cardiomyocyte hypertrophy clearly differed. Only a few of the genes significantly induced by PE were also increased by mechanical stretch to similar levels (upper lanes of Figure 2D). Yet, the majority of the PE-regulated transcripts were not significantly altered or even inversely regulated by stretch.
Thus, the microarray analyses revealed distinct differences in the gene expression pattern between stretch- and pharmacologically induced hypertrophy, suggesting that a stretch-specific gene program may exist.
Identification of Genes Specifically Induced by Biomechanical Stretch
To verify differential regulation of individual genes and ESTs in stretched cardiomyocytes we performed real-time PCR analyses. First, we confirmed the induction of several members of the "hypertrophic" gene program, for which an upregulation on prohypertrophic stimuli has been described before.22 The array data predicted the upregulation of ANF, BNP, FHL-1, BNIP3, and alpha skeletal actin (Table 1). We selected ANF, BNP, and FHL-1 for further real-time PCR analyses and confirmed significant upregulation of all 3 candidates (Figure 3). Biaxial stretch led to 5.1-fold induction of ANF (Figure 3A, P<0.001), whereas PE induced ANF 5.7-fold (P<0.001). Biomechanical stress resulted in 4.2-fold upregulation of BNP (Figure 3B, P<0.001), whereas PE induced BNP 2.9-fold (P<0.001). Likewise, the 4-and-a-half-LIM domains protein FHL-1 was significantly upregulated by both stretch (Figure 3C, 8.0-fold, P<0.01) and PE (9.0-fold, P<0.01).
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P<0.01, Next, we aimed to identify genes preferentially regulated in stretch-induced cardiomyocyte hypertrophy but not on pharmacological stimulation. Therefore, genes were chosen that fulfilled the following criteria: (1) The ratio of n-fold induction by stretch and n-fold induction by PE exceeded 2.0 and (2) induction by phenylephrine was less than 2.0x. A subset of 24 of 164 stretch-induced genes met these criteria and were thus considered to be candidate members of a stretch-specific gene program (summarized in Table 2). To further validate these findings, we carried out real-time PCR experiments for the genes with the strongest induction by stretch. All genes and ESTs examined by real-time PCR were found upregulated to a similar degree as predicted by the microarrays, confirming the accuracy of the latter experiment (Figure 4).
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Heat shock protein 70 (HSP 70, Figure 4A) displayed a marked regulation on stretch (20.9-fold, P<0.001 versus control), whereas PE only modestly induced HSP 70 (1.7-fold; P<0.001 versus stretch). Of note, HSP 70 as well as the protooncogene c-myc (Figure 4B, 3.0-fold induction by stretch, P<0.001) were among the first genes previously identified as stretch-responsive,23,24 confirming the reliability of our biomechanical stress model. In addition, several genes selectively induced by biomechanical stress could be identified, which have not been implicated in cardiomyocyte hypertrophy before: The positive regulator of cell cycle progression CKS2 (Cyclin dependent kinase regulatory subunit 2, Figure 4C) was found 5.8-fold upregulated by stretch (P<0.001 versus control), yet downregulated 0.6-fold by PE. Similarly, monoaminooxidase A (MaoA, Figure 4D) was induced 3.9-fold by stretch (P<0.001 versus control) but downregulated by PE. Moreover, the noncoding RNA VL30 (Figure 4E), implicated in gene regulation,25 was induced 5.2-fold by stretch (P<0.001 versus control) but not PE. Interestingly, several antioxidative and cardioprotective factors were markedly and stretch-specifically upregulated, such as metallothionein 1a (Figure 4F, 17.7-fold; P<0.001 versus control). Likewise, heme oxidase-1 (Hmox1, Figure 4G) also ranked among the most specifically regulated gene transcripts and was found induced by stretch 10.8-fold (P<0.05 versus control), whereas PE left the level of Hmox1 unchanged. The strongest upregulation was observed for the cytokine growth and differentiation factor 15 (GDF15 or Mic-1, Figure 4H), which was induced 24.8-fold through stretch (P<0.05 versus control), but not significantly regulated by phenylephrine. We further asked whether the highly induced genes GDF15 and heme oxidase-1 were upregulated on the protein level as well. Both GDF15 and Hmox1 indeed displayed a pronounced induction on stretch (Figure 4I and 4J), whereas phenylephrine failed to induce significant upregulation. In addition to the known genes described above, the microarray analyses also revealed several differentially regulated ESTs. Selective induction on biomechanical stress could be confirmed for all 4 tested transcripts (Figure S1), further supporting the notion of a stretch-specific gene program.
Finally, the expression levels of genes predicted to be downregulated on stretch were also determined by real-time RCR. Lipocalin 2, also named neutrophil gelatinase associated lipocalin, a protein induced in atherosclerosis and myocardial infarction,26 was found downregulated –86% by stretch (P<0.001) and –81% by PE (P<0.001 versus control, Figure 4K). The cardiac gap junction protein connexin-40 (Gja5) was decreased to 40% of the control on stretch (P<0.001) and to 18% by PE treatment (P<0.001 versus control, P<0.05 versus stretch, Figure 4L). Similarly, expression of the cell adhesion molecule VCAM1 (–59% by stretch, P<0.001; –76% by PE, P<0.001 versus control, Figure 4M), and phospholipase 2A (–91% by stretch, P<0.001; –80% by PE, P<0.001 versus control, P<0.05 versus stretch, Figure 4N) were markedly depressed both by stretch and PE. In contrast to the upregulated transcripts, none of the downregulated genes were specifically repressed by stretch.
Stretch-Specific Gene Expression Is Mediated by Ang II Receptor Signaling
To further confirm that the observed changes in gene expression were specific for biomechanical stress, we also tested whether other pharmacological stimulators of cardiomyocyte hypertrophy, such as ET1 and Ang II, were able to induce these genes. We thus treated NRVCMs with 100 nmol/L ET1 or 100 nmol/L Ang II, respectively. As a control, we again included NRVCMs stimulated with 50 µmol/L phenylephrine in this experiment. All 3 agents induced hypertrophy to a similar degree (Figure S2) and led to strong induction of members of the hypertrophic gene program, including ANF (Ang II: 5.0-fold, P<0.05; ET1: 7.6-fold, P<0.01; PE: 6.7-fold, P<0.01) and BNP (Ang II: 9.0-fold, P=0.03; ET1: 3.8-fold, p=n.s.; PE: 5.6-fold, P=0.01) (Figure 5A and 5B). Similar to phenylephrine, endothelin-1 did neither significantly induce GDF15 (ET-1: 1.2-fold, P=n.s.; PE: 1.2-fold, P=n.s.), nor heme oxidase I (ET-1: 1.0-fold, P=n.s.; PE: 0.9-fold, P=n.s.). In contrast, stimulation with Ang II caused a significant induction both of GDF15 (2.4-fold, P<0.01) and heme oxidase-1 (3.9-fold, P<0.001), supporting the notion that Ang II receptor signaling might be involved in the transduction of biomechanical stress19,27,28 (Figure 5C and 5D). Similarly, metallothionein-1 expression could not be stimulated by phenylephrine or endothelin-1, yet was significantly induced by Ang II (2.6-fold, P<0.001, Figure 5E). Several others of the previously identified stretch-dependent genes revealed a similar pattern, including cMyc, MaoA, VL30E, and CKS2 (Figure S3).
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Because even enriched neonatal cardiomyocytes are not completely devoid of other cell types, we also tested whether contaminating fibroblasts could have contributed to the observed stretch-specific gene expression. Thus, neonatal rat fibroblasts were expanded in vitro and subjected to stretch or treatment with 100 nmol/L Ang II. We again tested expression of several genes found to be upregulated in stretched cardiomyocytes, including GDF-15, HMOX1, and Mt1a. None of these transcripts were found to be significantly induced in fibroblasts (data not shown), suggesting that the observed stretch-dependent regulation is cardiomyocyte-specific.
Next, we were asking whether upregulation of the stretch-specific subset of genes is dependent on Ang II receptor 1a signaling. Thus, biaxially stretched neonatal cardiomyocytes were treated with the AT1-receptor blocker irbesartan (100 nmol/L) and analyzed for changes in the stretch-induced gene expression pattern (Figure 6). Irbesartan significantly attenuated stretch-induced cardiomyocyte hypertrophy (–60.1%, P<0.05; Figure 6A). Moreover, irbesartan significantly reduced the expression of BNP, GDF15, and heme oxidase-1 (Figure 6B through 6D). BNP expression was reduced by 38.3% (P<0.001, Figure 6B). The abundance of GDF15 mRNA was decreased by 65.9% (P<0.001, Figure 6C) on irbesartan treatment. Likewise, heme oxidase-1 expression was significantly blunted (–45.6%, P<0.001, Figure 6D) by irbesartan, whereas ANF (Figure 6E) and Mt1a induction (Figure 6F) were not altered. Taken together, these data suggest that at least in a subset of stretch-responsive genes, biomechanical stress is transduced via the Ang II receptor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biomechanical stress leads to the induction of cardiomyocyte hypertrophy which is accompanied by an increase in cell size, enhanced sarcomeric organization, and induction of the "fetal" gene program. Yet, it is unknown, whether the molecular signals activated by "stretch" differ from other hypertrophic stimuli, ie, endogenous neurohumoral agonists.
To our knowledge, the present study is the first genome-wide analysis of the gene expression pattern of stretched cardiomyocytes in comparison with pharmacological inducers of cardiomyocyte hypertrophy. Remarkably, only a total of 185 genes of more than 28 000 genes covered by the Affymetrix 230.2 chip were significantly regulated on stretch when adjusting the level of significance to a post-hoc probability of >99%. In contrast, phenlyephrine led to a broader alteration of gene expression with differential regulation of 450 genes when applying the same statistical criteria. Both biomechanical stretch and stimulation with the 
-adrenergic agonist phenylephrine led to a comparable degree of hypertrophy and the induction of the fetal gene program (including upregulation of ANF, BNP, FHL1, and several other genes), pointing to shared mechanisms in the development of hypertrophy. However, we also identified a subset of 24 genes with stretch-specific regulation, again suggesting that stretch-induced hypertrophy is mediated at least in part by a specific gene expression program.
We verified differential regulation of several candidates for stretch-specific regulation by real-time PCR and found that HSP70, metallothionein-1, heme oxidase I, and GDF15 displayed the highest stretch specificity (Figure 4a). Metallothionein-1 is an antioxidative protein, which renders cardiomyocytes resistant against oxidative stress.29 When overexpressed in a cardiac-specific manner, metallothionein is able to diminish the cardiotoxicity of doxorubicin.30 Furthermore, induction of endogenous metallothionein expression by administration of zinc protects mice against the development of diabetic or alcoholic cardiomyopathy.31,32 Similarly, upregulation of metallothionein mediates the cardioprotective effects of STAT3 in an ischemia-reperfusion model.33 One could thus hypothesize that metallothionein protects cardiomyocytes from reactive oxygen species induced by mechanical stress.34 Similarly, the highly induced heme oxidase I (HMOX1) is known as a cardioprotective factor as well. It degrades heme to biliverdin, iron, and carbon monoxide.35 Mice with heterozygous disruption of the HMOX1 gene are sensitized for ischemia-reperfusion injury,36 whereas overexpression of HMOX1 in the heart attenuates the detrimental effects of ischemia and reperfusion.37 HMOX1 is also sufficient to inhibit Ang II–induced cardiac hypertrophy by a bilirubin-mediated inhibition of reactive oxygen species.38 The p38 and extracellular signal-regulated kinase (ERK) 1/2 MAP kinases and the Calcineurin/NFAT pathway were identified as principal targets of the antihypertrophic actions of HMOX1.39 The induction of heme oxidase I by biomechanical stress could thus represent a counteracting mechanism to prevent pathological hypertrophy.
Another interesting finding is the strong and stretch-specific upregulation of the cytokine growth and differentiation factor 15 (GDF15) which belongs to the transforming growth factor (TGF)β superfamily.40 At baseline, GDF15 is only detectable in liver and placenta.40,41 Yet, it has recently been shown to be strongly inducible in the heart on pathological stimuli such as myocardial infarction and pressure overload.42,43 GDF15-deficient mice subjected to ischemia-reperfusion injury exhibited increased infarct sizes as well as an increase in apoptosis. Conversely, recombinant GDF15 protects cardiomyocytes from apoptosis via PI3K- and AKT-dependent mechanisms.43 Likewise, transgenic overexpression of GDF15 in the heart blunts pressure overload-induced hypertrophy, whereas GDF15(–/–) mice displayed more pronounced cardiac hypertrophy on aortic banding. In cultured myocytes, the antihypertrophic properties of GDF15 appeared to be mediated via SMAD-dependent pathways. These findings led the authors to propose that GDF15 is a cytokine released in an auto- or paracrine way that displays antihypertrophic and cardioprotective features. The experimental settings (remodeling after ischemia, aortic banding) in which GDF15 has been described to be induced42,43 are consistent with a higher wall tension resulting in increased biomechanical stress. Thus, these in vivo findings are consistent with our results that HMOX1 and GDF15 are preferentially regulated by stretch.
To further underscore the stretch-specificity of Mt1a/b, HMOX1, and GDF15, we also showed that not only phenylephrine but also endothelin-1 failed to significantly induce the transcription of these genes (Figure 5C). In contrast, stimulation with Ang II led to upregulation of all 3 transcripts. Stretch-induced induction of HMOX1 and GDF15 could be significantly blunted by irbesartan, suggesting that the angiotensin receptor plays a critical role in mediating biomechanical stress signals. However, we cannot exclude that endothelin-1 also contributes to stretch-dependent signaling,20 because we found c-myc to be induced by endothelin as well, albeit to a lesser degree than by Ang II. Moreover, signal transduction in response to phasic versus static stretch may differ, as previous reports have shown that phasic stretch is not only mediated by Ang II but also endothelin-1.44
How could the Ang II receptor signaling pathway modulate stretch-specific gene expression? How can the angiotensin-II pathway be distinct from the signaling pathways used by ET1 and PE, given that their receptors also couple to the Gq/11 G proteins? Interestingly, the angiotensin type 1 receptor (AT1R) does not only couple to Gq/11 or infrequently to Gi,45 but also to "unconventional" G protein–independent signaling mechanisms.46 The cytoplasmic domains of the AT1R interacts with multiple signaling proteins.47 Besides the G coupling site, the AT1R can bind to JAK2 in an agonist-dependent fashion leading to STAT1/2 activation.48 Similarly, the AT1R binds to phospholipase Cgamma1 presumably via SHP-2,49 to ATRAPs (AT1R associated proteins),50,51 and a novel protein called GLP, a GDP-/GTP exchange factor protein.52 Cardiac overexpression of an AT1R deficient for Gq/Gi coupling leads to exaggerated hypertrophy, yet less apoptosis and fibrosis on chronic Ang II stimulation compared with transgenic mice overexpressing the wild-type receptor.53 In an accompanying commentary, Rockman and coworkers46 speculated that GRKs and β-arrestins might be good candidates for the mediation of the Gq/Gi-independent AT1R-signals, because their intracellular functions mimic the effects of the AT1R deficient for G protein coupling.54 Very recently, Sadoshima’s group also found that mice transgenic for an Ang II type 1 receptor mutant without the ability to activate the EGF-receptor did neither develop cardiac hypertrophy nor fibrosis on Ang II infusion. This effect was observed despite a normal activation of the Gq G protein signaling cascade,55 again supporting the notion that G protein independent signaling properties of the AT1R may promote cell growth and protection against cell death and fibrosis.
Perspectives
In summary, we provide a comprehensive analysis of the gene expression profile of stretched cardiomyocytes. This profile could clearly be differentiated from the expression pattern induced by pharmacological agents such as phenylephrine and endothelin-1, suggesting the existence of a biomechanical stress-specific gene program. Moreover, we demonstrate that this gene program is mediated, at least in part, by angiotensin receptor signaling. We speculate that stretch-specific gene expression is evoked by activation of the angiotensin II type 1 receptor and transmitted downstream partly in a Gq/11-independent fashion.
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Acknowledgments |
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We are grateful to Urike Öhl and Jutta Krebs for excellent technical assistance. We also thank Sanofi-Aventis for providing the angiotensin receptor blocker Irbesartan.
Sources of Funding
D.F. was supported by the Young Investigator Program of the University of Heidelberg, N.F. and B.B. were supported by the Bundesministerium für Bildung und Forschung, Germany (NGFN2-Nationales Genomforschungsnetz). Part of this work was supported by a research grant from Sanofi-Aventis to D.F. and N.F.
Disclosures
None.
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Footnotes |
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The first 2 authors contributed equally to this study.
Received July 15, 2007; first decision August 12, 2007; accepted November 26, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Frey N, Olson EN. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol. 2003; 65: 45–79.[CrossRef][Medline] [Order article via Infotrieve]
2. Haunstetter A, Izumo S. Toward antiapoptosis as a new treatment modality. Circ Res. 2000; 86: 371–376.
3. Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the heart: a new therapeutic target? Circulation. 2004; 109: 1580–1589.
4. Devereux RB, Wachtell K, Gerdts E, Boman K, Nieminen MS, Papademetriou V, Rokkedal J, Harris K, Aurup P, Dahlof B. Prognostic significance of left ventricular mass change during treatment of hypertension. JAMA. 2004; 292: 2350–2356.
5. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med. 1990; 322: 1561–1566.[Abstract]
6. Bolognese L, Dellavesa P, Rossi L, Sarasso G, Bongo AS, Scianaro MC. Prognostic value of left ventricular mass in uncomplicated acute myocardial infarction and one-vessel coronary artery disease. Am J Cardiol. 1994; 73: 1–5.[CrossRef][Medline] [Order article via Infotrieve]
7. Dorn GW, 2nd, Robbins J, Sugden PH. Phenotyping hypertrophy: eschew obfuscation. Circ Res. 2003; 92: 1171–1175.
8. Frank D, Kuhn C, Katus HA, Frey N. The sarcomeric Z-disc: a nodal point in signalling and disease. J Mol Med. 2006; 84: 446–468.[CrossRef][Medline] [Order article via Infotrieve]
9. Knoll R, Hoshijima M, Hoffman HM, Person V, Lorenzen-Schmidt I, Bang ML, Hayashi T, Shiga N, Yasukawa H, Schaper W, McKenna W, Yokoyama M, Schork NJ, Omens JH, McCulloch AD, Kimura A, Gregorio CC, Poller W, Schaper J, Schultheiss HP, Chien KR. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell. 2002; 111: 943–955.[CrossRef][Medline] [Order article via Infotrieve]
10. Frey N, Barrientos T, Shelton JM, Frank D, Rutten H, Gehring D, Kuhn C, Lutz M, Rothermel B, Bassel-Duby R, Richardson JA, Katus HA, Hill JA, Olson EN. Mice lacking calsarcin-1 are sensitized to calcineurin signaling and show accelerated cardiomyopathy in response to pathological biomechanical stress. Nat Med. 2004; 10: 1336–1343.[CrossRef][Medline] [Order article via Infotrieve]
11. Brancaccio M, Fratta L, Notte A, Hirsch E, Poulet R, Guazzone S, De Acetis M, Vecchione C, Marino G, Altruda F, Silengo L, Tarone G, Lembo G. Melusin, a muscle-specific integrin beta1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload. Nat Med. 2003; 9: 68–75.[CrossRef][Medline] [Order article via Infotrieve]
12. Peng X, Kraus MS, Wei H, Shen TL, Pariaut R, Alcaraz A, Ji G, Cheng L, Yang Q, Kotlikoff MI, Chen J, Chien K, Gu H, Guan JL. Inactivation of focal adhesion kinase in cardiomyocytes promotes eccentric cardiac hypertrophy and fibrosis in mice. J Clin Invest. 2006; 116: 217–227.[CrossRef][Medline] [Order article via Infotrieve]
13. Yamazaki T, Komuro I, Kudoh S, Zou Y, Nagai R, Aikawa R, Uozumi H, Yazaki Y. Role of ion channels and exchangers in mechanical stretch-induced cardiomyocyte hypertrophy. Circ Res. 1998; 82: 430–437.
14. Chang J, Wasser JS, Cornelussen RN, Knowlton AA. Activation of heat-shock factor by stretch-activated channels in rat hearts. Circulation. 2001; 104: 209–214.
15. Kim CH, Cho YS, Chun YS, Park JW, Kim MS. Early expression of myocardial HIF-1alpha in response to mechanical stresses: regulation by stretch-activated channels and the phosphatidylinositol 3-kinase signaling pathway. Circ Res. 2002; 90: E25–E33.[CrossRef][Medline] [Order article via Infotrieve]
16. Komuro I, Yazaki Y. Control of cardiac gene expression by mechanical stress. Annu Rev Physiol. 1993; 55: 55–75.[CrossRef][Medline] [Order article via Infotrieve]
17. Sadoshima J, Izumo S. The cellular and molecular response of cardiac myocytes to mechanical stress. Annu Rev Physiol. 1997; 59: 551–571.[CrossRef][Medline] [Order article via Infotrieve]
18. O’Connell TD, Ishizaka S, Nakamura A, Swigart PM, Rodrigo MC, Simpson GL, Cotecchia S, Rokosh DG, Grossman W, Foster E, Simpson PC. The alpha(1A/C)- and alpha(1B)-adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse. J Clin Invest. 2003; 111: 1783–1791.[CrossRef][Medline] [Order article via Infotrieve]
19. Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993; 75: 977–984.[CrossRef][Medline] [Order article via Infotrieve]
20. Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Hiroi Y, Mizuno T, Maemura K, Kurihara H, Aikawa R, Takano H, Yazaki Y. Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. J Biol Chem. 1996; 271: 3221–3228.
21. Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem. 1999; 270: 41–49.[CrossRef][Medline] [Order article via Infotrieve]
22. Hoshijima M, Chien KR. Mixed signals in heart failure: cancer rules. J Clin Invest. 2002; 109: 849–855.[CrossRef][Medline] [Order article via Infotrieve]
23. Delcayre C, Samuel JL, Marotte F, Best-Belpomme M, Mercadier JJ, Rappaport L. Synthesis of stress proteins in rat cardiac myocytes 2–4 days after imposition of hemodynamic overload. J Clin Invest. 1988; 82: 460–468.[Medline] [Order article via Infotrieve]
24. Komuro I, Kaida T, Shibazaki Y, Kurabayashi M, Katoh Y, Hoh E, Takaku F, Yazaki Y. Stretching cardiac myocytes stimulates protooncogene expression. J Biol Chem. 1990; 265: 3595–3598.
25. Song X, Sun Y, Garen A. Roles of PSF protein and VL30 RNA in reversible gene regulation. Proc Natl Acad Sci U S A. 2005; 102: 12189–12193.
26. Hemdahl AL, Gabrielsen A, Zhu C, Eriksson P, Hedin U, Kastrup J, Thoren P, Hansson GK. Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction. Arterioscler Thromb Vasc Biol. 2006; 26: 136–142.
27. Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Mizuno T, Takano H, Hiroi Y, Ueki K, Tobe K, Kadowaki T, Nagai R, Yazaki Y. Angiotensin II partly mediates mechanical stress-induced cardiac hypertrophy. Circ Res. 1995; 77: 258–265.
28. Sadoshima J, Izumo S. Autocrine secretion of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Contrib Nephrol. 1996; 118: 214–221.[Medline] [Order article via Infotrieve]
29. Wang GW, Schuschke DA, Kang YJ. Metallothionein-overexpressing neonatal mouse cardiomyocytes are resistant to H2O2 toxicity. Am J Physiol. 1999; 276: H167–H175.[Medline] [Order article via Infotrieve]
30. Sun X, Zhou Z, Kang YJ. Attenuation of doxorubicin chronic toxicity in metallothionein-overexpressing transgenic mouse heart. Cancer Res. 2001; 61: 3382–3387.
31. Wang L, Zhou Z, Saari JT, Kang YJ. Alcohol-induced myocardial fibrosis in metallothionein-null mice: prevention by zinc supplementation. Am J Pathol. 2005; 167: 337–344.
32. Wang J, Song Y, Elsherif L, Song Z, Zhou G, Prabhu SD, Saari JT, Cai L. Cardiac metallothionein induction plays the major role in the prevention of diabetic cardiomyopathy by zinc supplementation. Circulation. 2006; 113: 544–554.
33. Oshima Y, Fujio Y, Nakanishi T, Itoh N, Yamamoto Y, Negoro S, Tanaka K, Kishimoto T, Kawase I, Azuma J. STAT3 mediates cardioprotection against ischemia/reperfusion injury through metallothionein induction in the heart. Cardiovasc Res. 2005; 65: 428–435.
34. Pimentel DR, Amin JK, Xiao L, Miller T, Viereck J, Oliver-Krasinski J, Baliga R, Wang J, Siwik DA, Singh K, Pagano P, Colucci WS, Sawyer DB. Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes. Circ Res. 2001; 89: 453–460.
35. Otterbein LE, Choi AM. Heme oxygenase: colors of defense against cellular stress. Am J Physiol Lung Cell Mol Physiol. 2000; 279: L1029–L1037.
36. Yoshida T, Maulik N, Ho YS, Alam J, Das DK. H(mox-1) constitutes an adaptive response to effect antioxidant cardioprotection: A study with transgenic mice heterozygous for targeted disruption of the Heme oxygenase-1 gene. Circulation. 2001; 103: 1695–1701.
37. Yet SF, Tian R, Layne MD, Wang ZY, Maemura K, Solovyeva M, Ith B, Melo LG, Zhang L, Ingwall JS, Dzau VJ, Lee ME, Perrella MA. Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice. Circ Res. 2001; 89: 168–173.
38. Hu CM, Chen YH, Chiang MT, Chau LY. Heme oxygenase-1 inhibits angiotensin II-induced cardiac hypertrophy in vitro and in vivo. Circulation. 2004; 110: 309–316.
39. Tongers J, Fiedler B, Konig D, Kempf T, Klein G, Heineke J, Kraft T, Gambaryan S, Lohmann SM, Drexler H, Wollert KC. Heme oxygenase-1 inhibition of MAP kinases, calcineurin/NFAT signaling, and hypertrophy in cardiac myocytes. Cardiovasc Res. 2004; 63: 545–552.
40. Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, Zhang HP, Donnellan M, Mahler S, Pryor K, Walsh BJ, Nicholson RC, Fairlie WD, Por SB, Robbins JM, Breit SN. MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci U S A. 1997; 94: 11514–11519.
41. Paralkar VM, Vail AL, Grasser WA, Brown TA, Xu H, Vukicevic S, Ke HZ, Qi H, Owen TA, Thompson DD. Cloning and characterization of a novel member of the transforming growth factor-beta/bone morphogenetic protein family. J Biol Chem. 1998; 273: 13760–13767.
42. Xu J, Kimball TR, Lorenz JN, Brown DA, Bauskin AR, Klevitsky R, Hewett TE, Breit SN, Molkentin JD. GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation. Circ Res. 2006; 98: 342–350.
43. Kempf T, Eden M, Strelau J, Naguib M, Willenbockel C, Tongers J, Heineke J, Kotlarz D, Xu J, Molkentin JD, Niessen HW, Drexler H, Wollert KC. The transforming growth factor-beta superfamily member growth-differentiation factor-15 protects the heart from ischemia/reperfusion injury. Circ Res. 2006; 98: 351–360.
44. Liang F, Gardner DG. Autocrine/paracrine determinants of strain-activated brain natriuretic paptide gene expression in cultured cardiomyocytes. J Biol Chem. 1998; 273: 14612–14619.
45. Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature. 2002; 415: 206–212.[CrossRef][Medline] [Order article via Infotrieve]
46. Rajagopal K, Lefkowitz RJ, Rockman HA. When 7 transmembrane receptors are not G protein-coupled receptors. J Clin Invest. 2005; 115: 2971–2974.[CrossRef][Medline] [Order article via Infotrieve]
47. Hunyady L, Catt KJ. Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol Endocrinol. 2006; 20: 953–970.
48. Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk BC, Delafontaine P, Bernstein KE. Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature. 1995; 375: 247–250.[CrossRef][Medline] [Order article via Infotrieve]
49. Venema RC, Venema VJ, Eaton DC, Marrero MB. Angiotensin II-induced tyrosine phosphorylation of signal transducers and activators of transcription 1 is regulated by Janus-activated kinase 2 and Fyn kinases and mitogen-activated protein kinase phosphatase 1. J Biol Chem. 1998; 273: 30795–30800.
50. Daviet L, Lehtonen JY, Tamura K, Griese DP, Horiuchi M, Dzau VJ. Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor. J Biol Chem. 1999; 274: 17058–17062.
51. Guo DF, Chenier I, Tardif V, Orlov SN, Inagami T. Type 1 angiotensin II receptor-associated protein ARAP1 binds and recycles the receptor to the plasma membrane. Biochem Biophys Res Commun. 2003; 310: 1254–1265.[CrossRef][Medline] [Order article via Infotrieve]
52. Guo DF, Tardif V, Ghelima K, Chan JS, Ingelfinger JR, Chen X, Chenier I. A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells. J Biol Chem. 2004; 279: 21109–21120.
53. Zhai P, Yamamoto M, Galeotti J, Liu J, Masurekar M, Thaisz J, Irie K, Holle E, Yu X, Kupershmidt S, Roden DM, Wagner T, Yatani A, Vatner DE, Vatner SF, Sadoshima J. Cardiac-specific overexpression of AT1 receptor mutant lacking G alpha q/G alpha i coupling causes hypertrophy and bradycardia in transgenic mice. J Clin Invest. 2005; 115: 3045–3056.[CrossRef][Medline] [Order article via Infotrieve]
54. Seta K, Nanamori M, Modrall JG, Neubig RR, Sadoshima J. AT1 receptor mutant lacking heterotrimeric G protein coupling activates the Src-Ras-ERK pathway without nuclear translocation of ERKs. J Biol Chem. 2002; 277: 9268–9277.
55. Zhai P, Galeotti J, Liu J, Holle E, Yu X, Wagner T, Sadoshima J. An angiotensin II type 1 receptor mutant lacking epidermal growth factor receptor transactivation does not induce angiotensin II-mediated cardiad hypertrophy. Circ Res. 2006; 99: 528–536.
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