Temporal Gene Expression Profiling Indicates Early Up-regulation of Interleukin-6 in Isoproterenol-induced Myocardial Necrosis in Rat

Abstract

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-κB 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.

Introduction

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.

Materials and Methods

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

Isoproterenol hydrochloride (Sigma, St. Louis, Missouri) in sterile water (n = 5 rats/group/time point) or sterile water (n = 3 rats/group/time point) was administered subcutaneously at the dosage of 0.5 mg/kg. Necropsies were performed at 9 time points postdosing: 0.5 hours, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 3 days, 7 days, and 14 days. At necropsy, the rats were anesthetized with isofluorane/O₂ anesthesia, and blood was collected from the retro-orbital sinus for clinical chemistry and hematology. The rats were exsanguinated by catheterization of the abdominal aorta, and a pneumothorax was induced.

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

Histology (hematoxylin and eosin [HE], phosphotungstic acid hematoxylin [PTAH], Masson’s trichrome), clinical chemistry, and hematology were evaluated at all time points. Hematology included erythrocyte count, hemoglobin, hematocrit, red cell distribution, platelets, platelet volume, and total and differential white blood cell count and was performed using the Abbott Cell-Dyn 3500 (Abbott Park, Illinois). Clinical chemistry included aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) and was performed on the Roche/Hitachi Modular Analytics system (Roche Diagnostics, Indianapolis, Indiana). Haptoglobin assessment was performed on the Roche/Hitachi Modular Analytics system using reagent from TriDelta Development Ltd. (Maynooth, Ireland). Evaluation of Il6 and Crp in the serum was performed using the Bender Medsystems and Kamiya Biomedical ELISA kits, respectively. Troponin I was evaluated using a Beckman Coulter Access 2 analyzer (Fullerton, California).

RNA Isolation, Quality Assessment, and cDNA Synthesis

Gene profiling was performed on the heart samples collected at 1 hour, 3 hours, 1 day, and 3 days. Total RNA was extracted from the heart tissues using the RNeasy Mini Kit (Qiagen, Valencia, California; Cat. #74104, 74106). Tissues were homogenized on the Tissue Lyser with the universal laboratory mixer-mill disruptor (Qiagen; Cat. #85210) using the protocol for isolation of total RNA from fibrous tissues with the DNase I treatment to remove any genomic DNA contamination. Total RNA quality was assessed on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California).

Conversion to cDNA, cRNA

Fifteen μg total RNA was converted into double-stranded cDNA by reverse transcription (Gibco Life Technologies, Grand Island, New York) using the T7-T24 primer (5′-GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG [dT24]). The double-stranded cDNA was cleaned up using the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, California). For conversion into copy RNA, the in vitro transcription (IVT) MEGAscriptTM T7 kit (Ambion) and biotinylated nucleotides (UTP and CTP) were used. The cRNA was cleaned and fragmented using the GeneChip Sample Cleanup Module.

Hybridization, Staining, and Image Analysis

Hybridization of fragmented IVT product to Rat Genome 230 2.0 Array and washing steps were performed as suggested by the manufacturer (Affymetrix). Each hybridized GeneChip array was scanned with an argon-ion laser scanner at 570 nm (Agilent/Affymetrix). Image analysis was done with the GCOS software (Affymetrix).

Affymetrix Data Analysis

Quality of the chips was assessed with a variety of analyses. Inspection of the residual images was used to identify potential problems with wet-lab procedures. Signal box plots and Gapdh ratios were used to assess RNA quality. In addition, multivariate procedures such as hierarchical clustering and principal-components analyses were used to assess the consistency of gene changes for each dose and time condition. All chips passed these quality assessments, and none were dropped from the analysis. Chips were preprocessed using robust multichip analysis (Irizarry et al., 2003) and quantile-quantile normalization. Normalized data were fit to a linear model with dose and time contrasts using the GLM procedure of SAS 9.1 (SAS Institute, Carey, North Carolina). The data were deposited in the database Gene Expression Omnibus of the National Center for Biotechnology Information under the accession number GSE7999 and GSM197604-197633.

Data Analysis

The threshold for statistical significance was set at p ≤ .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.

Quantitative RT-PCR (qPCR)

Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed for 19 genes at all time points. The gene transcripts for qRT-PCR analysis were selected to elucidate the kinetics of Il6 pathway activation as elements of the Il6 pathway (Il6, Il6st, Socs3), downstream targets of this pathway (A2m, Bcl2l1, Cebpb, Egr1, Figf, Il6st, Irf1, Junb, Lbp, Socs3, Timp1), markers of heart disease (Cav, Gucy1a3, Hand2, Nppa, Spp1), a histologic correlate of fibrosis (Col5a2), or as internal controls (Gapdh, Rn18s).

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:

$$\begin{array}{l}[(\text{Level\hspace{0.17em}of\hspace{0.17em}Target\hspace{0.17em}Gene\hspace{0.17em}}(\text{Treated})/\text{Level\hspace{0.17em}of\hspace{0.17em}Target\hspace{0.17em}Gene}\\ (\text{Vehicle\hspace{0.17em}Control})]/[\text{Level\hspace{0.17em}of\hspace{0.17em}}\text{Reference\hspace{0.17em}Gene}\\ (\text{Treated})/\text{Level\hspace{0.17em}of\hspace{0.17em}Reference\hspace{0.17em}Gene\hspace{0.17em}}(\text{Vehicle\hspace{0.17em}Control})].\end{array}$$

Results

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

Microarray analysis of RNA extracted from the heart showed that the transcriptomic profile was dependent on the time of evaluation. The number of statistically significant sequences was 1,866 1 hour postdosing and between 4,000 and 5,000 at the later time points (Supplemental Table 3). The direction of the fold changes of the qPCR data consistently reproduced that of the chip data (Supplemental Table 4). However, as anticipated, fold changes were more dramatic for the qPCR data than for the chip data.

Gene Ontology Analysis

Consistent with acute irreversible myocardial damage, the 1-hour time point was dominated by genes involved in cell death (Supplemental Table 5). At 3 hours, numerous genes involved in development and in response to the myocardial damage were represented. Overrepresentation of numerous genes involved in the development of the heart is consistent with the hypothesis that myocardial damage is followed by the initiation of a fetal gene program, a phenomenon that is characterized by re-expression of fetal structural genes, growth factors, and proto-oncogenes (Brand et al., 1993; Dunnick et al., 2006; Izumo et al., 1988; Masino et al., 2004; Mercadier et al., 1981). However, up-regulation of the archetypal cardiac fetal genes Myh7, Acta1, and Nppa occurred only at 1 day and 3 days.

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

The overlap in gene functions shared between the different time points was limited to a few functions at each time point (0 to 3 of the 20 most significant functions; Supplemental Table 6), which emphasizes the time-dependent nature of the transcriptomic profile after a single acute myocardial damage.

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)

The only overrepresented pathway at 1 hour was the interleukin 4 (Il4) signaling pathway, with an uncertain effect on the overall regulation of this pathway that is involved in the development of Th2 cells. However, the significant genes in this pathway are also involved in inflammation, metabolism, apoptosis, and response to stress, and hence, the significance of this pathway remains uncertain.

At 3 hours, pathway activation provided evidence of:

1. Irreversible myocardial damage assessed by over-representation of the p38 mitogen-activated protein kinase (MAPK) pathway. In our experience, over-representation of the p38 MAPK pathway is associated with acute myocardial necrosis. Similarly, evidence of apotosis and/or of the early stages of inflammation is provided by overrepresentation of the NF-κB, PDGF, Tgfb, and JAK/STAT signaling pathways and prostaglandin and leukotriene metabolism;

2. Prominent up-regulation of the Il6 pathway following prominent up-regulation of Il6 at 1 hour; and

3. Up-regulation of the PPAR pathway, which has been associated with myocardial ischemia/infarction (Sidhu and Kaski, 2001) and hypertrophy (Asakawa et al., 2002; Takano et al., 2003).

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.

Discussion

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

Pathway analysis identified overrepresentation of the p38 MAPK pathway at 3 hours. Similarly, this pathway was over-represented at 6 hours with 2 Hoffmann-La Roche proprietary compounds for which myocardial necrosis was identified at that time point but not with 3 other compounds not causing myocardial necrosis after a single administration, suggesting that its overrepresentation may be used as a biomarker of acute myocardial necrosis.

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

Specific genes, including Myh7, Acta1, and Nppa, are expressed in the fetal myocardium but not in the normal adult myocardium. Alterations in the expression of these genes in the adult myocardium is termed “reactivation of the fetal gene program” and has been reported in a variety of pathologic conditions of the myocardium, including pressure overload (Sharma et al., 2006), atrophy (Sharma et al., 2006), hypertrophy (Li et al., 2003), and mitochondrial damage in the myocardium (Dunnick et al., 2006). The current study confirmed the expression of these genes as a response to acute myocardial necrosis. However, these “fetal genes” are markers of myocardial malfunction rather than of acute myocardial infarction because of their relatively late onset of expression, and hence, they have limited value for the early and specific diagnosis of myocardial conditions.

Energy Balance

Energy use was markedly altered during isoproterenol-induced myocardial necrosis: many genes of the fatty acid metabolism pathway, that is, the major source of energy in the healthy myocardium (Ritchie and Delbridge, 2006; Sack et al., 1996), were down-regulated at 1 day and 3 days. Several other conditions have been reported to decrease fatty acid oxidation in the myocardium, including pressure overload (Ritchie and Delbridge, 2006), acute myocardial ischemia (Aasum et al., 2003), maladaptative myocardial hypertrophy following chronic myocardial infarction (Barger and Kelly, 1999; Strom et al., 2005), and the administration of PPARg agonists (Edgley et al., 2006). Although the net effect of all these conditions is to reduce the use of fatty acids by cardiomyocytes, these conditions differ with respect to which specific genes of the fatty-acid metabolism pathway are affected. For example, Ucp2, which was down-regulated in our experiment at 3 days and in rats with maladaptative myocardial hypertrophy (Strom et al., 2005), is up-regulated in the myocardium of rats dosed with isoproterenol through subcutaneous minipumps (Ishizawa et al., 2006) and in mice overexpressing β adrenergic receptors (Gaussin et al., 2003). A better understanding of the metabolism of the diseased heart will be gained as transcriptomic data become available for a larger number of compounds and conditions.

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

Troponin I is the archetypal biochemical marker of myocardial necrosis that correlated best with histology. It increased above range values 0.5 hours after isoproterenol administration, at a time when contraction bands were reliably identified by PTAH but not by HE. This observation was anticipated because cTnI is an integral element of the myofilament and because it is highly susceptible to proteolysis (Van Eyk et al., 1998). Hence, it was anticipated that the identification of contraction bands by PTAH would correlate well with the detection of cTnI. Indeed, immunolabeling for cTnI dramatically decreases in the areas of contraction bands at the 0.5-hour time point (L. Fritzky, unpublished observation). Evaluation of earlier time points (i.e., before 0.5-hour post isoproterenol dosing) is needed to confirm the correlation between contraction bands and release of cTnI.

Conclusion

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.