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CARDIOVASCULAR
Departments of Human Growth and Development (M.E., C.L.G., H.R.G.), Infectious Diseases (A.T.T.), Cardio-Thoracic Surgery (J.M.D.), and Internal Medicine (J.A.H.), University of Texas Southwestern Medical Center, Dallas, Texas
Received November 5, 2007; accepted December 17, 2007.
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6). As expected, ISO increased matrix metalloprotease (MMP) 2 and 9 activities, and administration of DOX reversed this effect. Transcriptional profiles of normal, ISO-, and ISO + DOX-treated mice were examined using microarrays, and the results were confirmed by real-time reverse transcriptase-polymerase chain reaction. Genes (206) were differentially expressed between normal and ISO mice that were reversibly altered between ISO- and ISO + DOX-treated mice, indicating their potential role in CH development and DOX-induced improvement. These genes included those involved in the regulation of cell proliferation and fate, stress, and immune responses, cytoskeleton and extracellular matrix organization, and cardiac-specific signal transduction. The overall gene expression profile suggested that MMP2/9 inactivation was not the only mechanism whereby DOX exerts its beneficial effects. Western blot analysis identified potential signaling events associated with CH, including up-regulation of endothelial differentiation sphingolipid G-protein-coupled receptor 1 receptor and activation of extracellular signal-regulated kinase, p38, and the transcription factor activating transcription factor-2, which were reduced after administration of DOX. These results suggest that DOX might be evaluated as a potential CH therapeutic and also provide potential signaling mechanisms to investigate in the context of CH phenotype development and regression.
). Pathological CH occurs as a consequence of biomechanical stress, such as prolonged arterial pressure overload or valvular heart disease. This form of CH is maladaptive because heart enlargement, although it abrogates the initial stimuli, is characterized by contractile dysfunction and a decrease in heart performance. Prolonged pathological hypertrophy is associated with a significant increase in the risk for progression to heart failure, ischemic heart disease, and sudden death (Kannel et al., 1969). This maladaptive process is presumably not beneficial due to the unfavorable prognosis associated with the disease and favorable outcome correlated with a reduction in mass of the left ventricle, irrespective of which type of treatment is applied (Mathew et al., 2001). Prognosis for patients diagnosed with cardiac hypertrophy is typically poor, and aside from drug therapies, the only "cure" at present is a heart transplant. Despite the availability of generally acceptable therapies, such as angiotensin-converting enzyme inhibitors and β blockers, CH remains a chronic and progressive process. More specific therapies are needed to prevent heart deterioration in individuals with hypertrophy and to reduce unwanted side effects associated with current medications.
It is not known whether a reduction in heart mass is responsible for the beneficial effects of current medications or whether it is a consequence of treatment, but it is accepted as a standard metric to assess the effect of therapies. The process of CH is complex and involves multiple cross-regulated signaling pathways (for review, see Frey and Olson, 2003) that culminate in massive alterations in myocardial architecture (Fard et al., 2000). Matrix metalloproteases (MMPs) are pivotal to this process as central mediators of cardiac remodeling in response to injury and/or cardiac wall stress. MMPs are abnormally increased in a wide variety of diseases, including CH (Wainwright, 2004), and their inhibition has been demonstrated as a potential therapeutic strategy for CH (Asakura et al., 2002; Chancey et al., 2002; Miura et al., 2003).
In this study, we used IRIDESCENT, a computational program that can detect previously unknown relationships between medical terms (e.g., small molecules, phenotypes, and genes) in Medline (Wren et al., 2004), to predict potential drug therapies for CH. The utility of IRIDESCENT as a drug discovery tool was previously confirmed by predicting and verifying the relationship between chlorpromazine and CH (Wren et al., 2004). In addition to several other candidates, doxycycline (DOX) was predicted as a potential therapy for pathological CH. The software identified DOX as a potential therapeutic partly because it was previously shown to generally inhibit MMPs (Griffin et al., 2005), which suggested to us that it might reduce a hypertrophic phenotype. DOX is a very well tolerated antibiotic and is also FDA approved, making it a particularly attractive candidate for CH therapy. Furthermore, DOX has been shown in ischemic-reperfusion studies to exert a protective effect when administered before the induction of myocardial infarction (Villarreal et al., 2003; Griffin et al., 2005). To evaluate the efficacy of DOX, mice were treated with the drug after induction of CH with the β-adrenergic agonist, isoproterenol (ISO), or TAB. We found that DOX indeed inhibited CH-associated MMP2/9 activation and attenuated ISO- and TAB-induced CH. The potential signaling mechanisms involved were investigated by analyzing the expression and activity of various molecules that could contribute to the development of cardiac hypertrophy. A putative signaling pathway was constructed based on these results, and it indicated that DOX perturbs ISO-induced up-regulation of endothelial differentiation sphingolipid G-protein-coupled receptor 1 (EDG1) receptor expression and activation of ERK, p38, and ATF-2.
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Materials and Methods |
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) and were approved by the University of Texas Southwestern Institutional Animal Care and Use Committee Guidelines (Dallas, TX). Experiments were performed using three mice per group for microarrays, six mice per group for aortic banding, and 10 mice per group for all others unless otherwise stated. Normal, untreated mice and mice that did not receive surgery (isoproterenol or aortic banding) but that did receive doxycycline served as negative controls. ISO-Induced CH. Eight-week-old male C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were challenged with ISO (Sigma-Aldrich, St. Louis, MO) at 40 mg/kg/day, administered s.c. via microosmotic pump insertion (ALZET 1007D; Braintree Scientific, Braintree, MA). Animals were anesthetized with isoflurane (1.5%) and oxygen (98.5%) with an animal ventilator (SurgiVet, Waukesha, WI). An incision (1 cm) was made on the back of each animal between the shoulder blades, and a micro-osmotic pump containing ISO dissolved in saline solution (0.9% NaCl) was inserted into the infrascapular s.c. tissue. Mice were sacrificed after 7 days of experimentation.
TAB-Induced CH. Increased pressure in the proximal aorta was induced by means of TAB, as described previously (Hill et al., 2000). Male mice (C57BL/6, 8 weeks old) were anesthetized with ketamine (100 mg/kg i.p.) plus xylazine (5 mg/kg i.p.), orally intubated with 20-gauge tubing, and ventilated (Harvard Apparatus Rodent Ventilator, model 687; Harvard Apparatus Inc., Holliston, MA) at 120 breaths/min (0.1-ml tidal volume). A 3-mm left-sided thoracotomy was performed at the second intercostal space. The transverse aortic arch was ligated (7-0 Prolene) between the innominate and left common carotid arteries with an overlying 27-gauge needle, and then the needle was removed, leaving a discrete region of stenosis. The chest was closed, and the left-sided pneumothorax was evacuated. Perioperative (24 h) and 1-week mortalities were <10% each. Mice were sacrificed after 21 days of experimentation.
Administration of DOX. DOX (6 mg/ml) (Sigma-Aldrich) was given in drinking water containing 5% sucrose, beginning immediately after surgery and through the remainder of the experiment. The solution was changed twice weekly because of the short half-life of DOX in water. Control animals were given 5% sucrose water without the drug.
Microarray Sample Preparation and Analysis. Animal hearts were rapidly removed and flushed with saline to remove residual blood. Total RNA was isolated from the left ventricles using TRIzol Reagent (Invitrogen, Carlsbad, CA), per the manufacturer's instructions, purified by phenol-chloroform extraction and ethanol precipitation, and 20 µg was reserved for microarray analysis. Samples were further processed, labeled, and hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) by the University of Texas Southwestern Medical Center Microarray Core. Data were analyzed using GeneSifter (VizX Labs, Seattle, WA), SAM (Stanford University, Palo Alto, CA), and Spotfire DecisionSite 8.2 (Spotfire, Inc., Somerville, MA).
One mouse heart was used for each array, and the experiment was performed in triplicate, generating a total of nine arrays. GeneSifter was used to perform RMA normalization, pair-wise comparisons of averaged signal intensity values, and Student's t test with Benjamini and Hochberg correction. Spotfire was used to perform pair-wise comparisons of the individual experiments, and two-class, unpaired comparisons were made using SAM. A gene was considered as significantly altered in expression if the average -fold change value was at least 2.0, the -fold change for each individual replicate comparison was at least 1.5, the corrected p value was less than 0.05, and the false discovery rate was less than 5%. In addition, genes that were altered between any two wild-type (WT) or control samples were removed because these alterations most likely represented normal variations among mice.
Histology. Mouse hearts were excised, fixed in 10% phosphate-buffered formalin for 48 h, and then embedded in paraffin. Cross-sectional slices along the minor axis were obtained with a microtome and then stained using Mayer's Hematoxylin and Eosin or Masson's Trichrome.
Real-Time Reverse Transcriptase-Polymerase Chain Reaction. Quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) was performed in the iCycler iQ (Bio-Rad, Hercules, CA) using SYBR Green I dye (QIAGEN, Valencia, CA), as described by the manufacturer. Each 25-µl reaction contained 100 ng of RNA, 2.5 µl of primers (Quantitect Primer Assays; QIAGEN), 12.5 µlof SYBR Green PCR master mix, and 0.25 µl of reverse transcriptase. A typical protocol included reverse transcription at 50°C for 30 min and a denaturation step at 95°C for 15 min followed by 35 cycles with 94°C denaturation for 15 s, 55°C annealing for 30 s, and 72°C extension for 30 s. Detection of the fluorescent product was performed at the end of the extension period at 60°C for 20 s. To confirm amplification specificity, the PCR products were subjected to a melting curve analysis. Negative controls containing water instead of RNA were concomitantly run to confirm that the samples were not cross-contaminated. Targets were normalized to reactions performed using Quantitect GAPDH primers (QIAGEN), and -fold change was determined using the comparative threshold method (Livak and Schmittgen, 2001).
MMP Activity. General MMP activity was measured using the Enzolyte 520 Generic MMP assay kit (AnaSpec, San Jose, CA). In brief, hearts were homogenized in 0.15 M NaCl, 20 mM ZnCl, 1.5 mM NaN3, and 0.01% Triton X-100. Samples were subsequently diluted in assay buffer to equal concentration (
45 µg), incubated with the FAM/QXL 520 FRET substrate at 37°C for 1 h in a 96-well plate, and the plate was read at 490 nm. 4-Aminophenylmercuric acetate-treated samples served as positive controls. Zymographies were performed using Ready-Gel zymogram gels (Bio-Rad) per the manufacturer's instructions. In brief, protein samples were diluted (1:2) in Bio-Rad zymogram sample buffer and resolved on 10% gelatin zymography gels at 100 V for 1.5 h. Sample proteases were then renatured in Bio-Rad renaturing solution for 30 min at room temperature, followed by overnight incubation in development solution at 37°C. Gels were then stained with 0.5% Coomassie Blue R-250 for 1 h and destained with 40% methanol and 10% acetic acid before visualizing. Clear bands represented activated proteases, the sizes of which were compared with a positive control (MMP-2/MMP-9 zymography standard; Millipore Bioscience Research Reagents, Temecula, CA). MMP-2/MMP-9 Inhibitor IV (Millipore Bioscience Research Reagents) was used as a negative control.
Immunoblotting. Left ventricles were homogenized in 100 mM Tris-HCl, pH 7.4, containing 15% glycerol, 2 mM EDTA, 2% SDS, and 0.1 mM phenylmethylsulfonylfluoride. Homogenates were heated at 95°C for 10 min, passed through a 23-gauge needle five times, and centrifuged at 12,000g for 10 min (Chen et al., 2001). Proteins (80 µg/ml) were resolved by 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto a nitrocellulose membrane (Amersham Hybond; GE Healthcare, Piscataway, NJ). Membranes were blocked with 5% milk (Bio-Rad) and probed with affinity-purified antibodies at 1:1000 dilution. Membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology Inc., Danvers, MA) and then exposed to chemiluminescence substrate (GE Healthcare). Affinity-purified anti-MMP2, anti-EDG-1, and anti-GAPDH antibodies were purchased from Santa Cruz Biochemicals (Santa Cruz, CA), and anti-phospho-p-38, anti-total p-38, anti-phospho-ERK, anti-total ERK, and anti-phospho-ATF-2 antibodies were obtained from Cell Signaling Technology.
Statistical Analysis. Values presented are expressed as mean ± S.E.M. All comparisons between groups were performed using a one-tailed Student's t test. Differences were considered statistically significant for p < 0.05.
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MMP Activity. General MMP activity was measured in the hearts of normal mice and those treated with ISO or ISO plus DOX. As shown in Fig. 2A, ISO induced general MMP activity, compared with untreated mice. This level of general MMP activity did not further increase after treatment with 4-aminophenylmercuric acetate, a known activator of MMPs and positive control. General MMP activity was abrogated when mice were treated with DOX and ISO, with activity levels similar to those of control mice. These data were reproducible and statistically significant. We also examined the protein level of MMP2, the activation of which is strongly correlated with adverse myocardial remodeling and increased heart size (Soini et al., 2001). As shown in Fig. 3, there was no appreciable increase in MMP2 protein levels in CH mice, with or without drug treatment. Likewise, MMP9 was not increased at the level of transcription, based on real-time RT-PCR results (data not shown). Zymographies indicated that the activity levels of both MMP2 (Fig. 2B) and MMP9 (Fig. 2C) were increased after ISO treatment and reduced with DOX administration. Based on microarray results and real-time RT-PCR (Tables 1 and 2), MMP3 was up-regulated in mice with ISO-induced CH (7.4-fold) and down-regulated after DOX treatment (2.9-fold). MMP3, which was previously shown to be substantially increased in dilated cardiomyopathy (Spinale et al., 2000), can degrade a wide range of extracellular proteins, as well as activate other MMPs. Although no other MMPs were found to be transcriptionally altered, based on microarray results and Western blot analysis of MMP2 (Fig. 3), an increase in general MMP activity in CH mice that was abrogated by DOX treatment was confirmed (Fig. 2).
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Effect of DOX on Gene Expression Profile. To assess the effect of DOX on cardiac gene expression, microarray analysis was performed on normal mice (WT) and mice with ISO-induced CH that were subsequently untreated (control) or treated with DOX. Based on the microarray analysis criteria (see Materials and Methods), there were 354 genes that were altered in expression between control and DOX-treated mice, 206 of which were specific to the disease (i.e., also altered between WT and control mice but in the opposite direction as that observed for control versus DOX-treated mice). In other words, genes that were up-regulated by ISO treatment (compared with WT mice) were down-regulated by DOX treatment (compared with control mice), and genes that were down-regulated by ISO treatment were up-regulated by DOX treatment. These 206 genes included those involved in the regulation of cell proliferation and fate, stress and immune responses, cytoskeletal and extracellular matrix organization, and cardiac-specific signal transduction. Eighteen of these genes were selected for verification by real-time RT-PCR (Table 1). Examples of genes previously reported to be associated with cardiac hypertrophy, which were up-regulated in mice with ISO-induced CH and down-regulated after DOX treatment, are shown in Table 2.
EDG1 was up-regulated (2.5-fold) in CH mice and down-regulated (2.4-fold) subsequent to DOX treatment (Table 2), which was confirmed at the protein level and Western blot analysis (Fig. 4). Likewise, TBRII is up-regulated (2.4-fold) in mice with ISO-induced CH and returned to normal after DOX treatment (Table 2). CDKN1A was also profoundly up-regulated (14.6-fold) in mice with ISO-induced CH and subsequently down-regulated (6.5-fold) by DOX. CDKN1A, MAP3k6, and Map3k8, which are activators of the c-Jun NH2-terminal kinase (JNK) pathway, were both up-regulated (5.90- and 2.68-fold, respectively) in CH mice, based on microarray analysis results (Table 2). In contrast, the major ventricular gap junction protein, connexin 43, was down-regulated (3.89-fold) in CH mice and returned to normal levels (up-regulated 3.61-fold) in response to DOX treatment.
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). Post-Translational Effects of DOX Administration. In addition to examination of DOX-induced transcriptional changes, we also investigated several proteins known to be associated with hypertrophy-related signaling pathways, as detailed in the literature. As shown in Fig. 4, the phosphorylated version of the mitogen-activated protein kinase (MAPK) ERK 1/2 and stress-associated p38 MAPK were up-regulated in mice with CH and reduced subsequent to DOX treatment. Alteration of these proteins was specific for the activated forms because antibodies that recognize both the phosphorylated and nonphosphorylated versions (total ERK and total p38) did not result in bands that differed substantially in size or intensity between conditions (Fig. 4). Phosphorylation of the downstream MAPK transcription factor ATF-2 was similarly increased in ISO mice and depressed after DOX treatment (Fig. 4). We also examined the protein levels of an upstream receptor, EDG-1, which, based on microarray results, was transcriptionally up-regulated in ISO mice and returned nearly to baseline after DOX treatment. Western blot analysis confirmed this trend, as shown in Fig. 4. Anti-GAPDH antibodies served as an internal control to ensure equal protein was loaded on the gels (Fig. 4).
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; Grenier et al., 2002; Griffin et al., 2005). This prompted us to test the drug in two different mouse models of CH. This study demonstrates the efficacy of DOX in attenuating CH in both ISO-induced and TAB-associated CH. Hypertrophy in animals that did not receive DOX treatment was characterized by an increase in heart wall thickness, especially on the left side, which was expected, given the increased workload of the left ventricle. This article reports for the first time that an FDA-approved antibiotic is effective in reducing the cardiac hypertrophy phenotype.
Human oral DOX therapy currently involves a dosage of approximately 3 mg/kg/day, given over a period of time ranging from 1 week to 60 days. The dosage used in this study (6 mg/ml in drinking water) is as much as 15 to 20 times higher than the human allometric equivalent (assuming mice drink approximately 3 ml/day on average). We chose this higher dosage to ensure that the effects observed would be reproducible and because we did not observe a statistically significant reduction in heart size when the human equivalent dose was used in preliminary experiments. In addition, this treatment regimen (6 mg/ml in drinking water) had been previously used in another study, which investigated the efficacy of DOX for the treatment of muscular dystrophy in mice (Davies et al., 2006). However, we did test the ability of 4 mg/ml treatments to reduce CH (data not shown), which recapitulated the effects of the higher dosage (6 mg/ml). Additional dose ranging experiments are required to firmly establish the minimal effective dose of DOX and to confirm its therapeutic potential to regress pre-established CH. In addition, a less acute form of hypertrophy, such as a spontaneous hypertensive rat model, which is more representative of slowly progressive heart disease, might better demonstrate potential beneficial effects of DOX at lower dosages. A different method of DOX administration might further improve the resolution of these experiments because delivery in water does not allow for precise control of dosage. Apart from its ability to inhibit MMP2/9, the underlying mechanism of DOX-induced attenuation of CH is not clear. The majority of genes that were altered in response to ISO treatment and returned to baseline levels after DOX administration were not obviously related to MMP expression or activity. This indicated to us that other signaling mechanisms might contribute to the beneficial effects of DOX.
Figure 5 details the proposed signaling events that occur in ISO mice that are perturbed by DOX treatment, based on microarray and Western blot analyses and on previously reported research. As shown, ISO could lead to transforming growth factor (TGF)β pathway signaling via TBRII up-regulation. When bound by its ligand (TGFβ), the TBRI subunit is recruited, binds to TBRII, and then recruits the adaptor molecule, SMAD. This is known to lead to subsequent activation of p38 and SP1 transcription factor. The TGFβ pathway, when activated, has also been shown to cause activation of TGF-β-activated protein kinase 1, which activates JNK. Our results indicate that ISO might cause JNK activation via an alternate route, engagement of the EDG1 receptor by its ligand sphingosine 1 phosphate (S1P), as shown in Fig. 5. Stimulation of the EDG1 receptor, which was up-regulated in CH mice and down-regulated subsequent to DOX treatment (Table 2), induces cardiac hypertrophy (Robert et al., 2001). The precise molecular events that occur between S1P engagement of EDG1 and cardiac hypertrophy are not known, but S1P has been shown to induce phosphorylation of stress-activated JNK protein kinase in vitro (Robert et al., 2001), which might be activated in response to ISO and reversed by DOX treatment, based on the results of this study. Yet another potential mechanism for JNK activation is phosphorylation of upstream MAPKs (Map3k6/Map3k8), each of which were shown to be up-regulated transcriptionally based on microarray results (Table 2). JNK activation leads to down-regulation of connexin 43 and activation of ATF-2 transcription factor, which causes up-regulation of MMPs, such as MMP2/9, and CDKN1A. The pathways shown have been associated with cardiac remodeling, based on multiple literature reports. DOX could perturb these signaling events by preventing up-regulation of TBRII and EDG1 (Fig. 5).
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; Pellieux et al., 2000). Upstream (Map3k6 and Map3k8) and downstream (ATF-2) signaling events detected in our study (Table 2; Fig. 4) indicate that JNK is probably activated by ISO and inhibited by DOX. At first, the activation of JNK in response to cardiac stress could be protective (Tachibana et al., 2006), but extended activation most likely leads to hypertrophy. For example, JNK activation contributes to remodeling of gap junctions that leads to loss of ventricular wall compliance and a profound decrease in epicardial conduction velocity (Petrich et al., 2004; Petrich and Wang, 2004) and was previously shown to cause reduction in connexin 43 levels (Petrich et al., 2004). Loss of connexin 43 has been shown to lead to ventricular dysfunction and cardiac arrhythmia, a lethal eventuality of progressive heart failure (Gutstein et al., 2001). Furthermore, patients with decompensated cardiac hypertrophy have been shown to have diminished levels of connexin 43, compared with patients with compensated hypertrophy, and connexin 43 has thus been proposed to significantly contribute to a maladaptive response to cardiac stress and subsequent heart failure (Kostin et al., 2004). However, we were unable to obtain evidence that JNK is produced in normal mice or in response to ISO or DOX treatment, by Western blot analysis. This could have been due to technical difficulties, or perhaps the JNK pathway is bypassed in our system. We are currently designing experiments to address this issue to delineate the role of JNK, if any, in ISO-induced CH and reversal of the CH phenotype by DOX.
TGF-β activity has also been linked to cardiac hypertrophy. For instance, TGF-β has been shown to mediate a hypertrophic response in cardiomyocytes in response to angiotensin II (Ikeuchi et al., 2004; Rosenkranz, 2004). TGF-β signaling involves binding to TGF-β receptor II (TBRII), which results in recruitment of TBRI and subsequent phosphorylation of SMAD proteins (Yu et al., 2002). TBRII has also been recently shown to link TGF-β signaling and MAPK pathways via activation of TGF-β-activated protein kinase 1 (Yu et al., 2002; Watkins et al., 2006), which occurs in response to pressure overload generated by aortic constriction and leads to cardiac hypertrophy and heart failure (Zhang et al., 2000). Down-regulation of the expression of TBRII in response to DOX treatment would thus presumably inhibit TGF-β-induced hypertrophy. Likewise, CDKN1A, which is up-regulated via SP1 and SMAD activation subsequent to TGF-β engagement of its receptor (Chuang et al., 2007), might contribute to ISO-induced hypertrophy because it is believed to be associated with cardiac pathophysiology and pressure overload (Ohki et al., 2004). Furthermore, DOX has been previously shown to down-regulate TGF-β signaling that leads to downstream activation of SMADs and MAPKs (ERK, p38, and JNK) and subsequent stimulation of MMPs (Li et al., 2004; Kim et al., 2005). ERK in particular is associated with increased cardiac cell growth in response to mechanical stress (Abeles et al., 2006), and like JNK, p38 has been shown to phosphorylate ATF-2, which contributes to cardiac hypertrophy (Fischer et al., 2001). Considered together, this study corroborates previous research and suggests additional signaling molecules that could be involved in CH and that are inhibited by DOX administration. An overview of these hypothesized signaling events is shown in Fig. 5.
In summary, we have demonstrated that the FDA-approved antibiotic, DOX, is effective in attenuating CH in mice and could therefore be investigated as a potential treatment for CH in humans. DOX is a generic drug, and testing would be relatively rapid and inexpensive compared with non-FDA-approved drug candidates. Dose studies could determine whether DOX is indeed suitable for human CH trials, and administration in mice suggests that it could additionally serve as a cardiovascular research tool.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CH, cardiac hypertrophy; MMP, matrix metalloproteinase; DOX, doxycycline; FDA, United States Food and Drug Administration; ISO, isoproterenol; TAB, transverse aortic banding; EDG1, endothelial differentiation sphingolipid G-protein-coupled receptor 1; ERK, extracellular signal-regulated kinase; ATF, activating transcription factor; WT, wild type; RT, reverse transcriptase; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TBR, TGF receptor; CDKN1A, cyclin-dependent kinase inhibitor 1A (p21); JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; TGF, transforming growth factor; S1P, sphingosine 1 phosphate.
Address correspondence to: Dr. Mounir Errami, Division of Translational Research, University of Texas Southwestern Medical Center, 2201 Inwood Rd., Dallas, TX 75390-9185. E-mail: mounir.errami{at}utsouthwestern.edu
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abeles D, Kwei S, Stavrakis G, Zhang Y, Wang ET, and Garcia-Cardena G (2006) Gene expression changes evoked in a venous segment exposed to arterial flow. J Vasc Surg 44: 863–870.[CrossRef][Medline]
Asakura M, Kitakaze M, Takashima S, Liao Y, Ishikura F, Yoshinaka T, Ohmoto H, Node K, Yoshino K, Ishiguro H, et al. (2002) Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat Med 8: 35–40.[CrossRef][Medline]
Chancey AL, Brower GL, Peterson JT, and Janicki JS (2002) Effects of matrix metalloproteinase inhibition on ventricular remodeling due to volume overload. Circulation 105: 1983–1988.
Chen Z, Chua CC, Ho YS, Hamdy RC, and Chua BH (2001) Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice. Am J Physiol Heart Circ Physiol 280: H2313–H2320.
Chuang T, Guh J, Chiou S, Chen H, Hung W, and Chuang L (2007) Sp1 and Smad3 are required for high glucose-induced p21WAF1 gene transcription in LLC-PK1 cells. J Cell Biochem 102: 1190–1201.[CrossRef][Medline]
Czarnowski D, Wojcik B, Langfort J, and Gorski J (1996) 5'-Nucleotidase and adenosine deaminase activities in hypertrophied rat heart: the effect of tachycardia. Rocz Akad Med Bialymst 41: 334–340.[Medline]
Davies JE, Berger Z, and Rubinsztein DC (2006) Oculopharyngeal muscular dystrophy: potential therapies for an aggregate-associated disorder. Int J Biochem Cell Biol 38: 1457–1462.[CrossRef][Medline]
Fard A, Wang CY, Takuma S, Skopicki HA, Pinsky DJ, Di Tullio MR, and Homma S (2000) Noninvasive assessment and necropsy validation of changes in left ventricular mass in ascending aortic banded mice. J Am Soc Echocardiogr 13: 582–587.[CrossRef][Medline]
Fischer TA, Ludwig S, Flory E, Gambaryan S, Singh K, Finn P, Pfeffer MA, Kelly RA, and Pfeffer JM (2001) Activation of cardiac c-Jun NH2-terminal kinases and p38-mitogen-activated protein kinases with abrupt changes in hemodynamic load. Hypertension 37: 1222–1228.
Frey N and Olson EN (2003) Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol 65: 45–79.[CrossRef][Medline]
Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, and Sorsa T (1998) Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv Dent Res 12: 12–26.
Grenier D, Plamondon P, Sorsa T, Lee HM, McNamara T, Ramamurthy NS, Golub LM, Teronen O, and Mayrand D (2002) Inhibition of proteolytic, serpinolytic, and progelatinase-b activation activities of periodontopathogens by doxycycline and the non-antimicrobial chemically modified tetracycline derivatives. J Periodontol 73: 79–85.[CrossRef][Medline]
Griffin MO, Jinno M, Miles LA, and Villarreal FJ (2005) Reduction of myocardial infarct size by doxycycline: a role for plasmin inhibition. Mol Cell Biochem 270: 1–11.[CrossRef][Medline]
Gutstein DE, Morley GE, and Fishman GI (2001) Conditional gene targeting of connexin43: exploring the consequences of gap junction remodeling in the heart. Cell Commun Adhes 8: 345–348.[Medline]
Hill JA, Karimi M, Kutschke W, Davisson RL, Zimmerman K, Wang Z, Kerber RE, and Weiss RM (2000) Cardiac hypertrophy is not a required compensatory response to short-term pressure overload. Circulation 101: 2863–2869.
Ikeuchi M, Tsutsui H, Shiomi T, Matsusaka H, Matsushima S, Wen J, Kubota T, and Takeshita A (2004) Inhibition of TGF-β signaling exacerbates early cardiac dysfunction but prevents late remodeling after infarction. Cardiovasc Res 64: 526–535.
Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC.
Kannel WB, Gordon T, and Offutt D (1969) Left ventricular hypertrophy by electrocardiogram. Prevalence, incidence, and mortality in the Framingham study. Ann Intern Med 71: 89–105.
Kim H, Luo L, Pflugfelder SC, and Li D (2005) Doxycycline inhibits TGF-β1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells. Invest Ophthalmol Vis Sci 46: 840–848.
Kostin S, Dammer S, Hein S, Klovekorn WP, Bauer EP, and Schaper J (2004) Connexin 43 expression and distribution in compensated and decompensated cardiac hypertrophy in patients with aortic stenosis. Cardiovasc Res 62: 426–436.
Li D, Chen Z, Song XJ, Luo L, and Pflugfelder SC (2004) Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells. Invest Ophthalmol Vis Sci 45: 4302–4311.
Li WG, Zaheer A, Coppey L, and Oskarsson HJ (1998) Activation of JNK in the remote myocardium after large myocardial infarction in rats. Biochem Biophys Res Commun 246: 816–820.[CrossRef][Medline]
Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–
CT method. Methods 25: 402–408.[CrossRef][Medline]
Mathew J, Sleight P, Lonn E, Johnstone D, Pogue J, Yi Q, Bosch J, Sussex B, Probstfield J, and Yusuf S (2001) Reduction of cardiovascular risk by regression of electrocardiographic markers of left ventricular hypertrophy by the angiotensin-converting enzyme inhibitor ramipril. Circulation 104: 1615–1621.
Miura S, Ohno I, Suzuki J, Suzuki K, Okada S, Okuyama A, Nawata J, Ikeda J, and Shirato K (2003) Inhibition of matrix metalloproteinases prevents cardiac hypertrophy induced by β-adrenergic stimulation in rats. J Cardiovasc Pharmacol 42: 174–181.[CrossRef][Medline]
Ohki R, Yamamoto K, Ueno S, Mano H, Misawa Y, Fuse K, Ikeda U, and Shimada K (2004) Transcriptional profile of genes induced in human atrial myocardium with pressure overload. Int J Cardiol 96: 381–387.[CrossRef][Medline]
Pellieux C, Sauthier T, Aubert JF, Brunner HR, and Pedrazzini T (2000) Angiotensin II-induced cardiac hypertrophy is associated with different mitogen-activated protein kinase activation in normotensive and hypertensive mice. J Hypertens 18: 1307–1317.[CrossRef][Medline]
Petrich BG and Wang Y (2004) Stress-activated MAP kinases in cardiac remodeling and heart failure; new insights from transgenic studies. Trends Cardiovasc Med 14: 50–55.[CrossRef][Medline]
Petrich BG, Eloff BC, Lerner DL, Kovacs A, Saffitz JE, Rosenbaum DS, and Wang Y (2004) Targeted activation of c-Jun N-terminal kinase in vivo induces restrictive cardiomyopathy and conduction defects. J Biol Chem 279: 15330–15338.
Raskoff WJ, Goldman S, and Cohn K (1976) The "athletic heart": prevalence and physiological significance of left ventricular enlargement in distance runners. J Am Med Assoc 236: 158–162.
Robert P, Tsui P, Laville MP, Livi GP, Sarau HM, Bril A, and Berrebi-Bertrand I (2001) EDG1 receptor stimulation leads to cardiac hypertrophy in rat neonatal myocytes. J Mol Cell Cardiol 33: 1589–1606.[CrossRef][Medline]
Rosenkranz S (2004) TGF-β1 and angiotensin networking in cardiac remodeling. Cardiovasc Res 63: 423–432.
Soini Y, Satta J, Maatta M, and Autio-Harmainen H (2001) Expression of MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart. J Pathol 194: 225–231.[CrossRef][Medline]
Spinale FG, Coker ML, Bond BR, and Zellner JL (2000) Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target. Cardiovasc Res 46: 225–238.
Tachibana H, Perrino C, Takaoka H, Davis RJ, Naga Prasad SV, and Rockman HA (2006) JNK1 is required to preserve cardiac function in the early response to pressure overload. Biochem Biophys Res Commun 343: 1060–1066.[CrossRef][Medline]
Villarreal FJ, Griffin M, Omens J, Dillmann W, Nguyen J, and Covell J (2003) Early short-term treatment with doxycycline modulates postinfarction left ventricular remodeling. Circulation 108: 1487–1492.
Wainwright CL (2004) Matrix metalloproteinases, oxidative stress and the acute response to acute myocardial ischaemia and reperfusion. Curr Opin Pharmacol 4: 132–138.[CrossRef][Medline]
Watkins SJ, Jonker L, and Arthur HM (2006) A direct interaction between TGFβ activated kinase 1 and the TGFβ type II receptor: implications for TGFβ signalling and cardiac hypertrophy. Cardiovasc Res 69: 432–439.
Wren JD, Bekeredjian R, Stewart JA, Shohet RV, and Garner HR (2004) Knowledge discovery by automated identification and ranking of implicit relationships. Bioinformatics 20: 389–398.
Yu L, Hebert MC, and Zhang YE (2002) TGF-β receptor-activated p38 MAP kinase mediates Smad-independent TGF-β responses. EMBO J 21: 3749–3759.[CrossRef][Medline]
Zhang D, Gaussin V, Taffet GE, Belaguli NS, Yamada M, Schwartz RJ, Michael LH, Overbeek PA, and Schneider MD (2000) TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice. Nat Med 6: 556–563.[CrossRef][Medline]
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