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Temporal Gene Expression Profiling Indicates Early Up-regulation of Interleukin-6 in Isoproterenol-induced Myocardial Necrosis in Rat
1 Hoffmann-La Roche Inc., Non-Clinical Drug Safety, Nutley, New Jersey, USA and Correspondence: Address correspondence to: Igor Mikaelian, Hoffmann La-Roche Inc., Non-Clinical Drug Safety, Bldg. 100/326, 340 Kingsland Street, Nutley, NJ, 07110; email: igor.mikaelian{at}roche.com.
Gene expression was evaluated in the myocardium of male Wistar rats after a single subcutaneous administration of 0.5 mg of isoproterenol, a β-adrenergic agonist that causes acute tachycardia with subsequent myocardial necrosis. Histology of the heart, clinical chemistry, and hematology were evaluated at 9 time points (0.5 hours to 14 days postinjection). Myocardial gene expression was evaluated at 4 time points (1 hour to 3 days). Contraction bands and loss of cross-striation were identified on phosphotungstic acid-hematoxylin-stained sections 0.5 hours postdosing. Plasma troponin I elevation was detected at 0.5 hours, peaked at 3 hours, and returned to baseline values at 3 days postdosing. Interleukin 6 (Il6) expression spiked at 1 to 3 hours and was followed by a short-lived, time-dependent dysregulation of its downstream targets. Concurrently and consistent with the kinetics of the histologic findings, many pathways indicative of necrosis/apoptosis (p38 mitogen-activated protein kinase [MAPK] signaling, NF-  B signaling) and adaptation to hypertension (PPAR signaling) were overrepresented at 3 hours. The 1-day and 3-day time points indicated an adaptive response, with down-regulation of the fatty acid metabolism pathway, up-regulation of the fetal gene program, and superimposed inflammation and repair at 3 days. These results suggest early involvement of Il6 in isoproterenol-induced myocardial necrosis and emphasize the value of early time points in transcriptomic studies.
Key Words: Affymetrix • gene expression • isoproterenol • interleukin 6 • myocardium • rat • transcriptomic Abbreviations: A2m, alpha-2-macroglobulin • ABI, Applied Biosystems • Acta1, actin, alpha 1, skeletal muscle • AR, adrenergic receptor • AST, aspartate aminotransferase • Bcl2, B-cell leukemia lymphoma 2 • Cav, caveolin • Cebpb, CCAAT enhancer binding protein (C EBP), beta • CK, creatine kinase • Col5a2, procollagen, type V, alpha 2 • Crp, C-reactive protein • Egr1, early growth response 1 • Figf, c-fos induced growth factor • Gapdh, glyceraldehyde-3-phosphate dehydrogenase • Gucy1a3, guanylate cyclase 1, soluble, alpha 3 • Hand2, heart and neural crest derivatives expressed transcript 2 • HE, hematoxylin and eosin • Hif1a, hypoxia inducible factor 1, alpha subunit • Hp, haptoglobin • Il4, interleukin 4 • Il6, interleukin 6 • Il6st, interleukin 6 signal transducer • JAK STAT, Janus kinase-signal transduction and activation • Junb, Jun-B oncogene • KO, knock-out • Lbp, lipopolysaccharide binding protein • LDH, lactate dehydrogenase • MAPK, mitogen-activated protein kinase • Myh7, myosin, heavy polypeptide 7, cardiac muscle, beta • NF-kB, nuclear factor-kappa B • Nppa, natriuretic peptide precursor type A • PDGF, platelet derived growth factor • PPAR, peroxisome proliferator activated receptor • PTAH, phosphotungstic acid hematoxylin • Pygm, muscle glycogen phosphorylase • Rn18s, 18S RNA • RT-PCR, reverse transcriptase polymerase chain reaction • Socs3, suppressor of cytokine signaling 3 • Spp1, secreted phosphoprotein 1 • TGF, transforming growth factor • Tgfb, transforming growth factor • Timp1, tissue inhibitor of metalloproteinase 1 • Ucp2, uncoupling protein 2 (mitochondrial, proton carrier)
Adrenergic receptors are G-protein-coupled receptors that are linked to adenylyl cyclase and use cAMP as a second messenger (Rohrer et al., 1999; Singh et al., 2000). The β1-adrenergic receptor (AR) predominates in the heart and the brain, while the β2-AR predominates in the lungs and the cerebellum. Consequently, β1-AR is the predominant AR subtype regulating heart rate and contractility, while β2-AR has a role in mediating smooth-muscle relaxation. Isoproterenol is a synthetic catecholamine and an agonist of β1-AR and β2-AR. The administration of a single high dose of isoproterenol is a well-established animal model of acute tachycardia and myocardial infarction (Rona et al., 1959). However, despite detailed studies on the chronology of catecholamine-induced myocardial necrosis (Maruffo, 1967), the mechanisms responsible for this form of cardiotoxicity are not known. Multiple mechanisms have been proposed. The most broadly accepted mechanism attributes myocardial necrosis to increased oxygen demand resulting from pharmacologically induced tachycardia (reviewed in Dhalla et al., 1992). Other hypotheses include coronary vasospasm, mitochondrial dysfunction, electrolyte alterations, accumulation of fatty acids and Ca++, formation of adenochromes, and induction of apoptosis. Numerous studies have reported alterations in the expression of individual or limited sets of genes in the myocardium after isoprotenerol administration. Some of these changes in myocardial gene expression share features with the fetal gene program observed in failing hearts (Boluyt et al., 1995; Lowes et al., 2002; Rothermel et al., 2001). However, a global transcriptional perspective on changes occurring during isoproterenol-induced myocardial infarction is needed. This study attempts to (1) correlate established histological and biochemical measurements of myocardial damage to gene expression data and (2) identify the key molecular events occurring in the early stages of myocardial infarction. Important results include the identification of early overexpression of interleukin 6 (Il6) and the exclusion of some of the hypotheses proposed earlier to account for isoproterenol-induced myocardial necrosis, including mitochondrial dysfunction, accumulation of fatty acid, and induction of apoptosis.
Animals Male Wistar rats (Crl:Wi[Han]) aged 7 weeks were obtained from Charles River Laboratories (Raleigh, North Carolina) and were acclimated 2 weeks before dosing. Rats were single-housed in polycarbonate solid-bottom cages in a controlled environment (temperature maintained at 22°C ± 2°C and humidity at 50% ± 20%) with ad libitum access to Purina Certified Rodent Diet #5002-9 (pellets) and reverse-osmosis filtered water. All experiments were conducted in accordance with the guidance of the Roche Animal Care and Use Committee.
Treatment and Sampling A sagittal section of the heart, running from the base to the apex and across the arch of the aorta, was made. The portion of the heart located in the right thorax was fixed in 10% neutral buffered formalin and processed for histology. The other half of the heart was stored in RNAlater (Ambion, Austin, Texas) at room temperature overnight and then at –70°C until RNA extraction. RNA extraction was performed on approximately 0.2 g of myocardial tissue originating from the apical portions of the free ventricular wall. This site was selected because it is the preferred area of isoproterenol-induced myocardial damage.
Histology and Clinical Pathology
RNA Isolation, Quality Assessment, and cDNA Synthesis
Conversion to cDNA, cRNA
Hybridization, Staining, and Image Analysis
Affymetrix Data Analysis
Data Analysis
Quantitative RT-PCR (qPCR) TaqMan Low Density Arrays (Applied Biosystems (ABI), Foster City, California; #4342247) with primers and probes for selected mRNAs were purchased from ABI. TaqMan primers and probe sequences contained on TaqMan Low Density Arrays are proprietary to ABI and therefore are not disclosed. The same RNA samples as those used for chip analysis were diluted to 1 µg per well and reverse transcribed to cDNAs. Aliquots of cDNA were mixed with ABI TaqMan PCR Master Mix (2X) and water, added to the 384-well Low Density Array, and then run in duplicates. Messenger RNA levels were quantified using TaqMan reagents and a kinetic PCR apparatus (ABI 7900HT). Levels of amplicons were normalized to Gapdh level from each well. The following equation was used to calculate fold induction:
Clinical Findings and Histopathology Three rats died within 1 hour of isoproterenol dosing. Absolute heart weight and heart weight relative to body or brain weight were not altered (data not shown). Histology of the hearts from treated animals revealed the typical findings associated with isoproterenol-induced myocardial necrosis (Bloom and Cancilla, 1969; Dhalla et al., 1992; Kutsuna, 1972): PTAH staining identified myocardial damage earlier than HE, with prominent myofiber contraction bands at the first necropsy time (i.e., at 0.5 hours; Figure 1; Supplemental Table 1 [For all supplemental material, go to http://tpx.sagepub.com/supplemental/.]). Following the contraction-bands stage, cardiomyocytes displayed cardiomyolysis that peaked between 6 hours and 1 day and was readily detectable on standard HE sections. Cardiomyolysis consisted of fragmentation of the sarcoplasm of cardiomyocytes with gradual phagocytosis of the debris by macrophages. The first inflammatory cells in the myocardium were rare eosinophils (0.5 to 3 hours) followed by a few neutrophils (3 hours to 3 days). However, the bulk of the inflammatory response consisted of macrophages that were first identified at 6 hours and peaked at 3 days (data not shown). Deposition of collagen fibrils was first detected at 3 days, with the deposition of collagen fibers starting at 7 days. These stages of fibrous tissue deposition were best visualized with Masson’s trichrome (data not shown).
Clinical Pathology and Hematology Elevation of cTnI was detected at 0.5 hours, peaked at 3 hours, and returned to baseline values by 3 days (Figure 2). Detailed cTnI profiles and other significantly altered clinical pathology and hematology parameters are presented in Supplemental Table 2.
Aspartate aminotransferase was approximately 2-fold greater than control values at 3 hours, peaked at about 3-fold greater than control values at 6 hours, was approximately 2-fold greater than control values at 12 hours, and then returned to control values for the rest of the experiment. Neutrophil counts started to increase above control values 1 hour after dosing, remained 3-fold to 4-fold greater than control values at 3 hours, 6 hours, and 12 hours, and returned to control values afterward. Monocyte counts were approximately 2-fold greater than control values at 3 hours and 6 hours and were otherwise within normal range. Serum Il6, haptoglobin, Crp, CK, and LDH were not altered at any time point.
Gene-by-Gene Analysis
Gene Ontology Analysis Consistent with the early accumulation of inflammatory cells in the myocardium, the 1-day time point was dominated by gene categories involved in inflammation. However, an adaptive response of the autonomic nervous system was also apparent with overrepresentation of categories of genes coding for this system. The 3-day time point was dominated by genes involved in tissue remodeling and inflammation. These categories correlate well with the histopathology that identified prominent numbers of macrophages and the early stages of collagen deposition at 3 days.
Function Analysis At 1 hour, the most significant functions were related to hypertension, which is a direct consequence of isoproterenol-induced tachycardia, and to apoptosis and the early stages of inflammation, which are an indication of acute myocardial damage as identified histologically. Genes involved in apoptosis were still overrepresented at 3 hours, but more categories related to recruitment of inflammatory cells were represented. Inflammation escalated at 1 day, with overrepresentation of genes involved in the oxidative stress, and early repair. Inflammation dominated the transcriptome at 3 days.
Pathway Analysis (Table 1)
At 3 hours, pathway activation provided evidence of:
The pathways overrepresented at 1 day and 3 days provided evidence of metabolic adaptation, with down-regulation of the metabolism of fatty acids, lysine, and valine; leucine and isoleucine degradation; and propanoate metabolism, all of which are reported to be altered during myocardial damage (Larkin et al., 2004; Pasque and Wechsler, 1984). Overrepresentation of the lysine pathway and N-glycan biosynthesis as well as B-cell receptor signaling were consistent with the early stages of fibrosis and inflammation. Quantitative RT-PCR was used to better monitor the expression of the key elements of the Il6 pathway (Table 2; Supplemental Figure 1). All the transcriptional target genes of the Il6 pathway evaluated in this study were significantly up-regulated at 1 or multiple time points. However, the expression of some genes peaked before (Egr1, Junb) or well after (A2m, Figf, and Lbp) the peak of Il6 expression. This suggests that factors other than Il6 control the expression of these genes. The expression of Socs3 was synchronous to that of Il6, which support the hypothesis that Socs3, a downstream target of Il6, is also a negative regulator of the Il6 pathway in the myocardium (Yasukawa et al., 2001). The expression of Bcl2, Cebpb, and Timp1 was synchronous or slightly delayed compared to Il6, which is consistent with the hypothesis that these genes are downstream targets of the Il6 pathway. Finally, the absence of massive alterations in the expression of Il6st, a gene coding for 1 of the 2 components of the Il6 receptor, suggests that Il6 up-regulation does not alter the expression of its own receptor.
Interleukin 6 Pathway This study identified a very early (1-hour to 3-hour) spike in the expression of the acute-phase gene Il6 with subsequent modulation of the expression of several genes downstream of Il6 and overrepresentation of the Il6 and JAK/STAT signaling pathways at 3 hours. Also, Il6 likely has an autocrine or paracrine effect rather than acting as a systemic mediator in myocardial necrosis, because high expression of Il6 in the myocardium did not translate into high levels of serum Il6. Interleukin 6 is predominantly produced by myocardial fibroblasts (Burger et al., 2001; Saito et al., 2000; Yin et al., 2003), although it has also been reported to be produced by cardiomyocytes (Chandrasekar et al., 1999; Yamauchi-Takihara et al., 1995) as a result of myocardial ischemia (Chandrasekar et al., 1999; Ikeda et al., 1992; Kukielka et al., 1995) or cardiotoxicity by beta-adrenergic agonists (Goebel et al., 2000). Binding of Il6 to membrane receptors activates the JAK/STAT and the MAPK pathways (Freed et al., 2003; Heinrich et al., 1998; Yin et al., 2003; Supplemental Figure 1). Importantly, these 2 pathways are overrepresented at 3 hours, subsequent to the spike of Il6 at 1 to 3 hours, supporting the hypothesis that Il6 up-regulation is an early and important phenomenon in isoproterenol-induced cardiotoxicity. However, other genes reported to have important roles in acute myocardial necrosis, such as Egr1 and Junb, peaked before Il6, although they are downstream targets of Il6. This indicates that the Il6 expression spike, although spectacular by its amplitude, may not be the single most significant factor in isoproterenol-induced myocardial necrosis. Indeed, the effects of Il6 on myocardial function are still under debate: several lines of evidence suggest that Il6 has detrimental effects on the damaged heart, because animals injected with anti-Il6 antibodies have reduced inflammation in several models of ischemia (Cuzzocrea et al., 1999; Kukielka et al., 1995) and Il6-KO mice have fewer histologic lesions and/or better survival than wild-type mice in some inflammatory models (Cuzzocrea et al., 1999; Eriksson et al., 2003). Yet, other studies support a protective function of Il6 in the myocardium, because cardiomyocyte-restricted Il6-KO mice are more susceptible to myocardial injury caused by doxorubicin or lipopolysaccharides (Jacoby et al., 2003) and because mediators that, similarly to Il6, bind to Il6st favor cardiomyocyte hypertrophy and survival (Yasukawa et al., 2001). Definitive determination of the role of Il6 in isoproterenol-induced cardiotoxicity would require comparing the effects of isoproterenol in wild-type and Il6-KO mice.
p38 MAPK Pathway p38 MAPKs are activated by a variety of environmental stresses and cytokines in the myocardium. The current status on the function of p38 MAPKs suggests that they promote cardiomyocyte apoptosis (Ma et al., 1999; Wold et al., 2005; Zhu et al., 1999), although some reports suggest that they prevent it (Craig et al., 2000). Preservation of the phosphorylated form of p38 MAPK for immunohistochemistry requires tissue perfusion with the fixative and hence was not attempted in this study.
Fetal Gene Program
Energy Balance The accumulation of fatty acids during isoproterenol-induced myocardial necrosis reported by others (Dhalla et al., 1992) is consistent with down-regulation of the fatty-acid oxidation pathway identified in our study. However, this study identified down-regulation of the fatty-acid oxidation pathway as a late phenomenon, and hence, it is the consequence rather than the cause of isoproterenol-induced myocardial necrosis. The net result of isoproterenol administration on the glycolytic metabolism of the myocardium is less clear: some important genes involved in glucose metabolism, such as Pygm (Li et al., 2003), were down-regulated, while some others, such as Hif1a (Kakinuma et al., 2001), were up-regulated. However, glucose metabolism may be better monitored biochemically than by transcriptomic methods. Primary mitochondrial damage has been proposed as the cause of isoproterenol-induced myocardial damage (reviewed in Dhalla et al., 1992). The results of our study do not support a primary role for mitochondrial damage in isoproterenol-induced cardiotoxicity, because the gene ontology category "mitochondrion" was significant only at 3 days, and also, there was no evidence of massive alterations of mitochondrial genes at earlier time points.
Correlation of Clinical Chemistry Data with Pathology
A considerable body of literature has been devoted to investigations of the pathophysiology of isoproterenol-induced cardiotoxicity; the current study proposes an important role for Il6, which supports the hypothesis that isoproterenol-induced cardiotoxicity parallels an acute myocardial ischemic event and excludes some of the hypotheses proposed earlier. Also, this study stresses the informative value of the early time points (1 hour and 3 hours) for transcriptomic profiling and shows that the later time points (1 day and 3 days) are reflective of adaptation. Future work should aim at testing the hypothesis of a central role for Il6 in isoproterenol myocardial necrosis.
Ms. Nadine S. Tare performed the Il6 assays. Dr. Isabelle Wells developed the Hoffmann-La Roche Inc. proprietary software "GoSubtree." Drs. Rani Sellers, David Brewster, Martin Lamb, Michael Linn, Baolian Liu, Steven Stefanski, and Bernie Wagner provided constructive criticism in the preparation and interpretation of this study.
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Abstract
Introduction
.05 and an absolute fold value 
1.5 for all analyses. Gene ontology analysis was performed using the Hoffmann-La Roche Inc. proprietary software GoSubTree (Wells I., Basel, Switzerland). The software Ingenuity Pathway Analysis (Redwood City, California) was used for Pathway analysis. 
