INTRODUCTION

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RECENT FINDINGS from a number of laboratories have elucidated the cellular and molecular responses to myocardial overload and injury (2, 18, 30). However, much of this work has been accomplished using in vitro models of cardiac myocyte hypertrophy and failure, which have the disadvantage of removing the myocytes from the accompanying milieu of circulating hormones and other trophic factors, neural regulation, and the paracrine regulatory effects of cardiac nonmyocyte cells. In vivo models have demonstrated the complexity of these molecular responses, which vary with the nature, severity, and duration of the insult and affect both cardiac myocytes and nonmyocytes (4, 8, 16, 60). However, much of this work has involved analyses of myocardial tissue, and when studies have been performed on specific cell types, the focus has been on cardiac myocytes.

Much less is known of the responses of cardiac nonmyocytes, which comprise up to 70% of the cell population of the ventricular myocardium (31, 61), to these pathological conditions. This heterogeneous group of cells consists primarily of fibroblasts but also includes endothelial cells, smooth muscle cells, and macrophages. Collectively, these cells are responsible for both the production and maintenance of the support network of the heart, the cardiac interstitium (51). In addition to performing a structural role in the heart, however, it has become increasingly evident that cardiac nonmyocytes are also involved in the regulation of myocardial growth and gene expression via paracrine products, such as cytokines and other growth factors. Nonmyocytes can modulate myocardial function via these products and through alterations in their elaborated extracellular matrix (27, 51, 52, 58).

After myocardial infarction, the heart undergoes a remodeling process that is characterized by hypertrophy of the surviving myocytes and hyperplasia of the nonmyocytes (1, 19, 52). Although perhaps these responses initially serve an adaptive function, ultimately these alterations are often accompanied by depressed contractile function. During this remodeling process, the expression of several growth factors and cytokines is activated (12, 15, 48). For example, increased plasma levels, as well as local myocardial production, of several proinflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor- (TNF-), have been observed in patients early after experiencing acute myocardial infarction (11, 21, 32, 45). There is evidence that the circulating levels of cytokines may correlate with myocardial ischemia and dysfunction and explain, at least in part, the subsequent alterations in myocyte and nonmyocyte growth. Specifically, several cytokines have been reported to regulate cardiac myocyte growth, contractile protein synthesis, fibroblast proliferation, and extracellular matrix gene expression (3, 14, 29, 35, 46, 47, 55). These observations suggest that growth factors and cytokines may be important modulators in the postmyocardial infarction remodeling process, including infarction-associated inflammation, cardiac hypertrophy, fibrosis of myocardium, and cardiac dysfunction.

Although the potential importance of cytokine induction in the postinfarct period is recognized, there is limited information on the cell-specific changes in growth factor, cytokine, and collagen gene expression after myocardial infarction. Most information about the inflammatory cytokine expression after myocardial infarction has been obtained from measurements of circulating blood levels in patients early after experiencing acute myocardial infarction. Other data have been derived primarily from myocardial tissue samples, which contain a mixture of both myocytes and interstitial cells. Furthermore, the time course of the alterations in cytokine expression by specific myocardial cells has not been evaluated. Therefore, we undertook this study to examine the expression of important growth factor, cytokine, and extracellular matrix genes in the nonmyocyte compartment of the heart following myocardial infarction in the rat.

	METHODS

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Myocardial infarction and heart failure model. The rat coronary ligation model was used to induce myocardial infarction and heart failure (37). Adult male Sprague-Dawley rats were anesthetized with halothane, a left thoracotomy was performed, the pericardium was opened, and the heart was briefly exteriorized. The left coronary artery was ligated 1-2 mm from its origin with a 7-0 silk suture. Then the heart was returned to its normal position with the pericardium left open, and the thorax was closed. The same procedure was followed for sham-operated control animals, but the coronary ligature was left untied. After the operation, rats were housed in 2-animal Plexiglas cages, given regular food and water, and subsequently studied after 1 day and 1 or 6 wk.

The electrocardiogram (ECG) was used on selected rats with extensive infarction of the left ventricle. Six standard and limb leads and three precordial leads were evaluated. Only animals with large Q waves in leads I, aVL, and V5 were utilized. Extensive infarction was also confirmed by gross examination at the time of euthansia. The age at the time of infarction was selected so that at the end of the protocol, all rats were 14 wk of age. The overall mortality was 55% for the infarction groups and 4% for the sham groups.

Immediately before being euthanized, the rats were anesthetized with Innovar (1.3 ml/kg im), the right external carotid artery was cannulated with a 2-Fr microtip pressure transducer catheter (model PR 407, Millar Instruments, Houston, TX), and the right jugular vein was cannulated with saline-filled polyethylene-50 tubing connected to a pressure transducer (Spectromed, Gould, Cleveland, OH). After arterial blood pressures were obtained, the catheters were advanced into the left and right ventricles, respectively, and left ventricular pressures, pressure time derivatives (dP/dt), and right ventricular pressures were measured. To be certain that the experimental cohort consisted of animals with large infarcts, only animals with left ventricular end-diastolic pressures 16 mmHg were included from the infarct groups. Out of the 22 surviving infarcted rats, 18 (83%) met both the ECG and hemodynamic criteria and comprised the infarct groups. Several of the rats that comprised the final sham and infarct groups were the same animals as utilized in a previous report that examined the changes in gene expression in myocytes (60).

Cell isolation. Immediately after the hemodynamic measurements, the rats were administered heparin, and the heart from each rats was excised and perfused by a modified Langendorff technique at 37°C. Myocytes and nonmyocytes were enzymatically isolated with collagenase (43). Oxygenated Krebs-Henseleit (KH) (pH 7.4) solution, containing (in mM) 138 NaCl, 4.7 KCl, 1.5 CaCl₂, 1.2 MgSO₄, 10 glucose, 10 pyruvate, 5 HEPES, and 20 µ/l insulin, was used for the perfusion. After 5 min of equilibration at a perfusion pressure of 70 cmH₂O, the perfusion was switched to a constant-flow system at 8 ml/min, and the perfusate was changed to a nominally Ca²⁺-free KH solution for 5 min. Then 1 mg/ml collagenase B (Boehringer Mannheim, Indianapolis, IN) and 30 µM CaCl₂ were added to the Ca²⁺-free KH solution. After 30-40 min of collagenase perfusion, the heart was removed from the cannula, and the left ventricle, including the interventricular septum, was separated. The undigested infarcted myocardium and scar tissue were removed, and the remaining noninfarcted left ventricle was minced in oxygenated fresh Kraftbrühe buffer (pH 7.2), containing (in mM) 70 K-glutamate, 25 KCl, 10 KH₂PO₄, 10 oxalic acid, 10 taurine, 11 glucose, 2 pyruvate, 2 K-ATP, 2 phosphocreatine, 10 HEPES, and 5 MgCl₂. The resulting cell suspension was filtered through a 200-µm metal mesh to remove tissue debris and centrifuged at 50 g for 2× 5 min to remove the myocyte pellets. Then the nonmyocytes were collected by centrifugation of the supernatant at 150 g for 5 min. Myocyte contamination, examined under microscope by hematoxylin-eosin staining, was <1%. Myocytes isolated from the same hearts were sized by a Coulter Multisizer (Coulter Electronics, Hialeah, FL) (42), in order to evaluate cellular hypertrophy following myocardial infarction.

Northern blots. Total RNA was extracted from the isolated nonmyocytes by the guanidinium thiocyanate phenol-chloroform method (6) and quantified by absorbency at 260 nm. Fifteen-microgram nonmyocyte total RNA from each heart was size-fractionated by 1.2% agarose-formaldehyde gel electrophoresis, transferred to nylon membranes (Schleicher & Schuell, Keene, NH), and hybridized with ³²P-labeled specific probes for the mRNAs of interest. cDNA probes for IL-1 and TNF- were 1,400 and 1,100 bp EcoR I fragments of murine IL-1 and TNF- cDNAs, respectively (Genentech, South San Francisco, CA). The cDNA probe for IL-6 was a 900-bp BamH I/Pst I fragment of rat IL-6 cDNA (ATCC number 63087), and the cDNA probes for rat TGF-1 and mouse TGF-3 were kindly provided by Drs. A. Roberts and M. Sporn (National Cancer Institute, Bethesda, MD) (26). The probe for vascular endothelial growth factor (VEGF) was a 570-bp Xba/Bam fragment of a mouse VEGF cDNA obtained from Dr. L. T. Williams (University of California, San Francisco, CA). The probe for type I collagen was a 1,300-bp Pst/Bam fragment of a rat a₁(I) procollagen cDNA from Dr. D. Rowe (University of Connecticut, Farmington, CT) (9), and the probe for type III collagen was a 500-bp Xba I fragment of a mouse a₁(type III) procollagen cDNA from Dr. Y. Yamada (National Institute of Dental Research, Bethesda, MD) (23). All probes were labeled by random priming (Random Primed DNA Labeling Kit, Boehringer Mannheim) with [³²P]dCTP or antisense cRNA transcripts produced by [³²P]UTP labeling using MAXIscript T3/T7 in vitro transcription kits (Ambion, Austin, TX). For cDNA probes (IL-1, IL-6, TNF-, VEGF, and type I and type III collagens), blots were hybridized at 42°C for 18 h and washed 2× for 15 min in 6× standard saline citrate (SSC) plus 0.1% sodium dodecyl sulfate (SDS), then 2× 15 min with 2× SSC plus 0.1% SDS at 42°C. For cRNA probes (TGF-1, TGF-3, and 18S rRNA), blots were hybridized at 65°C for 18 h and washed 2× for 15 min in 150 mM NaPO₄ plus 1% SDS, then 2× for 15 min in 50 mM NaPO₄ plus 0.33% SDS at 65°C. Blots were stripped in 0.1× SSC and 0.1% SDS after each hybridization and reprobed in the sequence of: IL-1, collagen I, TNF-, collagen III, IL-6, VEGF, TGF-1, TGF-3, and 18S RNA. All blots were exposed to film (Kodak, Biomax-MR, Rochester, NY) at 70°C with an intensifying screen. Autoradiograms were quantified by scanning densitometry (Scan Analysis, Biosoft, Ferguson, MO), and signals were normalized to the respective 18S rRNA signal.

Statistics. The mRNA levels of each gene of interest were normalized to 18S rRNA in the same nonmyocyte RNA sample and were expressed as ratios of infarct to sham animals. The statistical significance of group (sham vs. infarct rats) and time-related (1 day and 1 and 6 wk) differences on the expression of each gene of interest and of hemodynamic variables were determined by two-factor analyzes of variance and Fisher's multiple comparison procedure. Linear regression analysis was used to test for the correlation between collagen mRNA expression and the levels of several cytokine-growth factor mRNAs that might play a role in its regulation. A P value <0.05 was taken as the threshold for statistical significance. All results are presented as means ± SE.

	RESULTS

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Hemodynamics. As required by the protocol, all rats in the infarct groups had extensive left ventricular infarction by visual inspection and Q waves and elevated left ventricular end-diastolic pressures. Although heart weights are not available because of the need to commence heart perfusion and digestion immediately, Table 1 shows the hemodynamic measurements and lung weights. Left ventricular dysfunction was evidenced at each time point in the infarct group by elevations of the left ventricular end-diastolic pressure and reductions in peak dP/dt. Right atrial pressures were also elevated in all infarct groups, as was right ventricular systolic pressure the 6-wk time point. This latter finding, together with the significantly increased lung weights in the 6-wk postinfarction rats, demonstrates the presence of chronic congestive heart failure.

Myocyte volume increased in the infarct group only at the 6-wk time point (4.2 ± 0.1 vs. 3.2 ± 0.1 × 10⁴ µm, infarct vs. sham, P < 0.01).

Inflammatory cytokines. Figure 1A shows representative Northern blots demonstrating the mRNA expression of inflammatory cytokines, IL-1, IL-6, and TNF- in freshly isolated nonmyocytes after myocardial infarction. mRNA signals for IL-6 and TNF- were barely detectable by Northern blotting in nonmyocytes from the sham-operated rats at any time point but increased postinfarction, particularly at the 1-wk time point. A strong signal for IL-1 was found in both groups at all time points, including an increase in expression in sham-operated animals, which could reflect a response to the cell isolation procedure. Figure 1, B-D, summarizes the data for these mRNAs, expressed as ratios of mRNA levels in infarct and sham rats. IL-1 expression was increased in the infarct group at 1 day and 1 wk (1.3 ± 0.1- and 1.3 ± 0.1-fold, respectively, both P < 0.01). IL-6 and TNF- became detectable at 1 day postinfarct, but the increase was not statistically significant until the 1-wk time point (2.1 ± 0.1- and 2.1 ± 0.2-fold, respectively, both P < 0.01). There were no significant differences in cytokine mRNA expression at 6 wk, although clearly increased signals for IL-6 and TNF- were present in two of six infarct rats at this time point (Fig. 1A), indicating the possibility of prolonged enhanced expression of cytokines after infarction in some animals. To confirm the cell-specific expression of these cytokines in nonmyocytes, RNA extracted from adult myocytes isolated from sham and infarcted hearts was subjected to Northern blotting and hybridized with the same cytokine cDNA probes.

View larger version (84K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   View larger version (30K): [in this window] [in a new window]   View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effects of myocardial infarction on proinflammatory cytokine mRNA expression. A: autoradiographs of representative Northern blots showing interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-, and 18S rRNA expression in nonmyocytes (NMC) from one sham (SH) and two infarcted (MI) rats at 3 time points. B, C, and D: bar graphs showing NMC mRNA levels for IL-1 (B), IL-6 (C), and TNF- (D). mRNA levels were normalized to level of 18S rRNA and expressed as ratio of MI to sham. IL-1 mRNA expression increased significantly after MI at day 1 and remained elevated at 1 wk but was back to baseline after 6 wk. IL-6 and TNF- expression were also elevated at 1 wk after MI (the changes at 1 day were not statistically significant). It is noteworthy that in 2 of 6 rats, mRNA levels for all 3 cytokines in nonmyocytes were still elevated at 6 wk after MI (A). In this and succeeding figures, each bar represents the ratio of mRNA in MI group/sham group (means ± SE) for 6 rats (n = 5 for 6-wk sham group). ** P < 0.01, MI vs. sham.

Growth factors. mRNA expression of three growth factors (TGF-1, TGF-3, and VEGF) were examined in the nonmyocytes. As shown in Fig. 2, compared with sham groups, mRNA expression of TGF-1 and TGF-3 in nonmyocytes was unchanged at 1 day, significantly increased at 1 wk (1.4 ± 0.2- and 1.5 ± 0.3-fold, infarct vs. sham, respectively, both P < 0.05), and returned to baseline by 6 wk after infarction. There were no significant changes in nonmyocyte VEGF mRNA expression at any of the observed time points (data not shown). This result contrasts with our previously reported finding of increased myocyte expression of VEGF at both 1 and 6 wk postinfarction (60).

View larger version (32K): [in this window] [in a new window]   View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effects of MI on tranforming growth factor (TGF)-1 and TGF-3 mRNA expression. Bar graphs showing nonmyocyte mRNA levels for TGF-1 (A) and TGF-3 (B). Data are presented as MI-to-sham ratios of normalized mRNA. mRNA expression for both cytokines was increased at 1 wk but returned to baseline by 6 wk after MI. ** P < 0.01, MI vs. shams.

Collagens. As shown in Fig. 3, A-C, compared with sham groups, both type I and type III collagen mRNA expression increased significantly in nonmyocytes 1 wk after myocardial infarction (2.2 ± 0.4- and 1.9 ± 0.1-fold, respectively, both, P < 0.01), with no difference from the sham groups either at 1 day or 6 wk after infarction.

View larger version (73K): [in this window] [in a new window]   View larger version (27K): [in this window] [in a new window]   View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effects of MI on collagen (Col) mRNA expression. A: autoradiographs of representative Northern blots showing collagen type I and type III mRNA and 18S mRNA expression in nonmyocytes from one SH and two MI rats at 3 treatment points. B and C: bar graphs showing nonmyocyte mRNA levels for collagen type I (B) and type III (C). mRNA levels were normalized to level of 18S rRNA. Expressed as a ratio of MI to shams, both type I and type III collagen mRNA expression increased markedly in nonmyocyte 1 wk after MI, with no difference from shams either 1 day or 6 wk after MI. ** P < 0.01, MI vs. sham.

Correlations of cytokine mRNA expression with cardiac function and collagen expression. There were no significant correlations between the relative increase in expression of the measured cytokines and growth factors and cardiac function, as assessed by left ventricular end-diastolic pressure. However, the potential detection of such relationships was limited by the inclusion of only rats selected for having elevated pressure levels.

To determine whether there were significant relationships between increased expression of type I and type III collagen mRNAs and the changes in cytokine and growth factor expression, linear regression analyses were performed. As shown in Fig. 4, type III collagen mRNA expression was significantly correlated with each of the cytokines and growth factors examined, with r values ranging from 0.78 (IL-6) to 0.53 (IL-1). Significant, but somewhat weaker, relationships were observed with collagen I (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Correlation of nonmyocyte collagen mRNA expression with expression of cytokines. Scattergram showing correlation and linear regression of type III collagen mRNA expression with several cytokines. Points obtained at all 3 post-MI time points were combined in this analysis. There were significant positive correlations between the mRNA for type III collagen and each of the cytokines, suggesting the possibility that these cytokines may play a role in regulating collagen expression. Similar relationships between type I collagen and the same cytokines were observed (data not shown). ** P < 0.01.

	DISCUSSION

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Major findings of present study. The primary finding of this study is the presence of distinct time and cell-specific changes in growth factor-cytokine gene expression in the period following acute extensive myocardial infarction associated with heart failure in the rat. Specifically, 1) the mRNAs encoding IL-6 and TNF- are induced within 24 h from undetectable levels in nonmyocyte cells and tend to persist for several weeks; the mRNA for IL-1, although expressed in the sham groups, is also upregulated. 2) TGF-1 and TGF-3 mRNA expression are elevated in nonmyocytes by 1 wk postinfarction and return to baseline by 6 wk. However, there is no alteration in nonmyocyte VEGF expression. 3) These findings differ from our previous findings in cardiac myocytes in the same model, in which we found no increase in TGF-1 and TGF-3, but increased mRNA for VEGF 1 and 6 wk postinfarction (60). 4) The mRNA expression of the main components of the extracellular matrix (type I and type III collagens) is increased 1 wk after myocardial infarction in nonmyocytes. 5) A possible regulatory role of nonmyocyte-produced growth factors-cytokines on extracellular matrix responses postinfarction is suggested by the similar time course and quantitative relationships between their expression and that of collagens.

Expression of proinflammatory cytokines postinfarction. These findings complement and extend prior observations in both patients and experimental animals. Increased plasma levels of IL-1, IL-6, and TNF- have been reported in patients with acute myocardial infarction (11, 21, 32, 45). Similarly, in rats after coronary ligation, a biphasic induction of mRNAs for these proinflammatory cytokines was observed in the postischemic myocardium within hours and at 7 days after infarction (15). This early increase in cytokine levels may reflect the inflammatory response induced by ischemia and/or infarction, and the subsequent rise may reflect healing after myocyte necrosis or a response to heart failure. Our results are consistent with these previous studies and further indicate that the myocardial expression of inflammatory cytokines is cell specific and may persist for several weeks after myocardial infarction in some animals. Because most previous studies have focused on the early stages of myocardial ischemia or infarction, there are no reports of prolonged cytokine expression after myocardial infarction. However, more persistent cytokine expression is observed in myocarditis and cardiomyopathy (24, 39) and has also been observed in congestive heart failure (22, 50). Taken together, these observations suggest a possible role for cytokines in myocardial injury and cardiac adaption/maladaption (38).

Interest and knowledge about the role of cytokines in various disease states and pathophysiological responses have increased recently (38). It is known that under various conditions, cytokines can be expressed in several types of cells, such as macrophages, lymphocytes, endothelial cells, fibroblasts, and smooth muscle cells. With respect to the heart, evidence for myocyte expression of both IL-6 and TNF- have been reported recently (17, 57). However, the specific cells involved have not been determined conclusively. By isolating myocytes and nonmyocytes from noninfarcted myocardium, we have demonstrated that after myocardial infarction the expression of IL-1, IL-6, and TNF- mRNAs are clearly induced in the nonmyocyte fraction. Preliminary results suggest that expression of these cytokines in myocyte fractions from the same model is increased to a much smaller extent, if at all, although alterations in other myocyte-specific genes were demonstrated (60). Because cytokines are thought to play important roles in modulating cardiac function and remodeling (see below) (17, 23, 38, 57), nonmyocyte expression of cytokines suggests possible autocrine and paracrine functions for cardiac nonmyocytes.

Changes in growth factor mRNA expression. Increased mRNA for both TGF-1 and TGF-3 were also observed in the nonmyocyte fraction 1 wk after myocardial infarction. Previous reports have suggested that TGF-1 and TGF-3 mRNAs can be expressed in both myocytes and nonmyocytes under certain circumstances (4, 44, 49, 58). In one study, increased expression of TGF-1 was observed in myocytes in the border zones of the infarct 24-48 h after infarction by immunostaining (48). Positive staining was also seen in neutrophils in the myocardial interstitium at the same time. Data from later time points were not provided. Our results indicate that nonmyocytes are a major cellular source of the increased TGF-1 and TGF-3 expression 1 wk after infarction. Increased myocyte TGF-1 and TGF-3 mRNA expression has also been reported in rats with pressure-overload-induced hypertrophy (4, 36), although these investigators also noted that the nonmyocytes express the majority of the TGF-1 and TGF-3 mRNA. It should also be recognized that there are important differences between the pathophysiology of chronic pressure overload and the postinfarction state, which could result in significantly different cellular and molecular responses. Indeed, there are many potential stimuli to postinfarction remodeling, including acute necrosis and inflammation, regional increases in wall stress, volume overload, and continuing myocardial ischemia, all of which may affect the expression of cytokines and growth factors.

Changes in extracellular matrix mRNA expression. Fibrosis and other alterations in the extracellular matrix are important aspects of the postinfarction-remodeling process affecting both the infarcted and noninfarcted myocardium, and these changes are thought to play a role in the evolution of left ventricular dysfunction (24, 54, 56). Types I and III collagen are the major components of the cardiac extracellular matrix and appear to account for most of the myocardial fibrosis following injury (44). Furthermore, their expression is regulated differently in response to different pathophysiological states. Increased collagen expression has been seen in pressure-overload hypertrophy (5, 13) but not in volume overload (13), whereas collagen expression is decreased in thyroid hormone-induced hypertrophy (59). In the present study, expression of mRNAs coding both type I and type III collagens was significantly increased in nonmyocytes at the 1-wk time point after myocardial infarction. This time course is consistent with observations of others in noninfarcted cardiac tissue (7) and resembles those in pressure-overload hypertrophy (5, 13).

Possible significance of changes in nonmyocyte cytokine and growth factor expression. Although myocardial infarction may result in substantial loss of functional myocardium and lead to acute cardiac decompensation, it is now well recognized that the subsequent changes in the noninfarcted myocardium play an important role in the longer term (10, 36). This process, which involves changes in myocytes (hypertrophy, dysfunction) and in the extracellular matrix, has been termed remodeling. Although the importance of this remodeling process is recognized, less is known of the cellular and molecular processes that regulate it. In that regard, considerable attention has been devoted to a potential role for cytokines and growth factors that are known to be increased postinfarction (3, 33, 35, 46, 47, 53). Much of this work has been based on changes in myocardial tissue or has focused on alterations in myocyte gene expression and function, but the importance of alterations in other cell types in the myocardium has been increasingly recognized (52, 53). Our results show that the expression of these substances is significantly, and perhaps predominantly, affected in nonmyocytes in the postinfarction period.

Possible effects of increased expression of cytokines and growth factors in the postinfarct period include increased fibrosis, myocyte hypertrophy, and myocyte dysfunction (25). Although our results do not permit determination of the consequences of the altered expression of these substances, the time course of these changes is at least consistent with some of these effects. We found that the increases in collagen gene expression after myocardial infarction coincided or followed those of the proinflammatory cytokines and TGF-1 and TGF-3 and were correlated with their expression.

Myocyte hypertrophy is another important feature of the postinfarction remodeling process. In the present study, myocyte size increased significantly between the 1- and 6-wk time points, indicating the development of significant cellular hypertrophy. This period follows the significant increase in nonmyocyte cytokine and TGF-1 and TGF-3 mRNA expression observed at 1 wk. IL-1 and TGF-1 are known hypertrophic stimuli to myocytes (28, 33, 34, 40). It is likely that these cytokines and growth factors produced by nonmyocytes play an important role in postinfarct myocyte hypertrophy and contractile protein expression.

Limitations. An important aspect of this study was the examination of changes in gene expression specific to the nonmyocyte compartment. Although it is likely that some contamination by myocytes occurs in this preparation, the absent or minimal changes in the expression of many of these mRNAs in the myocyte fraction suggests that the observed increases occur predominantly in the nonmyocytes (60). Similarly, the known ability of the cell isolation process to induce many of the cytokine genes studied (as a result of endotoxin in the collagenase, mechanical perturbation, artificial substrate, and free radicals in the culture medium) would lead to an underestimation of the actual magnitude of the differences in gene expression between the infarcted and sham-operated animals.

Because of the requirement for perfusing the heart during the isolation procedure, it was not possible to examine cells from the noninfarcted contralateral wall, peri-infarct border zone, and infarcted myocardium separately. It is unlikely that cells from the infarcted area contribute much to these findings, since this tissue was largely undigested. The border zone is relatively small in volume compared with the contralateral wall, but it is not possible to estimate the proportion of the signals of interest that were derived from this area. Similarly, it is impossible to determine what proportion of the mRNA responses are derived from fibroblasts (the predominant nonmyocyte cells in normal myocardium) or inflammatory cells.

Finally, this study only examined changes in gene expression at the transcriptional level, and therefore other processes that might effect protein levels (translation blockade, altered degradation) were not evaluated.

Summary and implications In the postinfarction period, there are significant increases in mRNA expression of several proinflammatory cytokines and growth factors by cardiac nonmyocyte cells isolated from the noninfarcted myocardium. These observations highlight the importance of examining cell-specific changes in the expression of these substances and in elucidating the role of cell-to-cell interactions within the myocardium. It is reasonable to speculate that the increases in postinfarction nonmyocyte cytokine and growth factor expression observed in the present study, as well as others that were not evaluated, may play important autocrine and paracrine functions in regulating the alterations in myocardial extracellular matrix and in myocyte growth. These alterations in nonmyocyte gene expression may be an important target for therapy and indeed may be involved in the mechanism by which drugs such as angiotensin-converting enzyme inhibitors favorably impact on the postinfarction remodeling process.