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(Circulation. 1997;95:1247-1252.)
© 1997 American Heart Association, Inc.

Articles

Tumor Necrosis Factor- Provokes a Hypertrophic Growth Response in Adult Cardiac Myocytes

Tomoyuki Yokoyama, MD; Masayuki Nakano, MD, PhD; John L. Bednarczyk, PhD; Bradley W. McIntyre, PhD; Mark Entman, MD; Douglas L. Mann, MD

the Cardiology (T.Y., M.N., D.L.M.) and Cardiovascular Sciences (M.E.) Sections of the Department of Medicine, Veterans Administration Medical Center and Baylor College of Medicine, and Department of Immunology, University of Texas M.D. Anderson Center (J.L.B., B.W.M.), Houston.

	Abstract

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Background Tumor necrosis factor- (TNF-) is a pleiotropic cytokine with a broad range of concentration-dependent effects. The recent observation that TNF- is expressed by the cardiac myocyte after certain forms of stress suggests that TNF- might contribute to the maintenance of normal tissue homeostasis after environmental injury. Accordingly, the purpose of this study was to examine the effects of TNF- on protein synthesis in cultured adult cardiac myocytes.

Methods and Results Cultured adult feline cardiac myocytes were stimulated with 10 to 1000 U/mL TNF- to examine the effects of this cytokine on the rate of protein synthesis and degradation. Stimulation with TNF- led to an accelerated rate of general protein synthesis and a time-dependent decrease in protein degradation in adult cardiac myocytes. The specificity of these findings was demonstrated by studies in which the effects of TNF- on protein synthesis were blocked by a neutralizing anti–TNF- antibody as well as studies in which TNF-–conditioned medium had no effect on protein synthesis in myocytes. In addition to the TNF-–induced increase in the general protein synthesis, stimulation with TNF- led to a 2.4-fold increase in net actin protein synthesis and a 3.3-fold increase in net myosin heavy chain synthesis. Finally, the effects of TNF- on adult cardiac myocytes were shown to be dependent on cell-substrate interaction, suggesting that the cell signaling pathways used by TNF- are dependent on a preserved interaction between cell integrins and the extracellular matrix.

Conclusions The observation that TNF- provokes a hypertrophic growth response in cardiac myocytes suggests that TNF- may play an important role in myocardial homeostasis after environmental stress.

Key Words: myocytes • peptides • cells • hypertrophy • growth substances

	Introduction

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The ability of the heart to undergo repair, remodeling, and hypertrophy after hemodynamic overloading or myocardial infarction ultimately determines whether the heart will decompensate and fail or whether instead it will maintain preserved function. Although the mechanisms that mediate tissue homeostasis after cardiac injury remain poorly understood, it is becoming increasingly clear that myocardial and nonmyocardial cell types residing within the mammalian heart both produce and respond to a variety of soluble protein factors that are capable of regulating myocardial growth.^{1 2 3 4 5} Relevant to the present discussion is the observation that TNF-, a proinflammatory cytokine with a broad range of pleiotropic effects, is expressed de novo by cardiac myocytes after certain forms of stress.^{6 7 8} Although the precise biological role for stress-induced TNF- expression within the heart is unknown, the observation that TNF- plays an important role in modulating homeostasis in a variety of different tissues^{9 10 11} raised the interesting possibility that TNF- might play an important role in regulating myocardial homeostasis in the adult heart as well.

Although at least two reports have examined the effects of TNF- on myocardial protein synthesis and degradation in vivo,^{12 13} it is probably not possible to isolate and precisely define the actions of this cytokine in vivo, given the complex effects of TNF- on serum levels of cortisol, insulin, and norepinephrine,^{12 14} any or all of which may have independent effects on myocardial growth.^{12 14} Moreover, it is unclear from the above studies whether TNF- influenced protein synthesis in cardiac myocytes and/or nonmyocardial cells, which may have different responses to peptide growth factors and cytokines.¹⁵ Accordingly, the purpose of this study was to provide a focused approach to determining the protein-synthetic effects of TNF- in isolated adult cardiac myocytes. The results of the present study suggest that concentrations of TNF- that are available to the cardiac myocyte after environmental stress are sufficient to provoke a hypertrophic growth response in cardiac myocytes.

	Methods

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Cardiac Myocyte Isolation and Culture
The methods for isolating adult feline cardiac myocytes, the characteristics of the cell culture system, and the cell culture conditions used here have all been described in considerable detail.^{16 17 18} The only exception to these methods is that all medium supplements were omitted from the M199 culture medium beginning on day 1 in culture. Endotoxin-free 0.1% HSA was always used as the carrier protein for the recombinant human TNF- (Genzyme) used in all experiments; accordingly, 0.1% HSA was always used as the control (diluent) solution. All animals were used in accordance with the guidelines of the Animal Care and Use Committee at Baylor College of Medicine and in compliance with the rules governing animal use published by the National Institutes of Health.

Cardiac Myocyte Evaluation
Evaluation of the Culture System
To determine whether the concentrations of TNF- used here would result in changes in cell viability, we examined the effects of TNF- on two indices of myocyte viability: the number of rod-shaped cardiac myocytes remaining adherent in culture and the percentage of rod-shaped cells that excluded trypan blue.¹⁸ Freshly isolated cells (5x10⁴/mL) were plated onto laminin-coated (24.4 nmol/L [20 µg/mL]) culture dishes that were inscribed with 2x2-mm² grids. The cells were permitted to attach to the laminin substrate for 24 hours, then washed two times with medium M199 to remove unattached or partially attached cells, and then exposed to fresh medium M199. Beginning on day 1 in culture, the cells were treated with diluent (0.1% HSA) or TNF- (200 U/mL). The total numbers of rod-shaped cells adherent to the laminin-coated surface were enumerated serially on days 1 through 3 by counting of the absolute cell number in numbered grid areas. To be certain that only cells that were firmly adherent to the culture dish were counted, the myocyte cultures were washed gently to remove unattached or partially attached cells before the cells were enumerated. The culture medium was replaced daily with fresh medium M199, TNF- (200 U/mL), and diluent (0.1% HSA). Results were expressed as the absolute number of rod-shaped cells/mm² remaining adherent on days 1 through 3 in culture. As an additional index of cell viability, we also examined the percentage of rod-shaped cells that excluded 0.4% trypan blue in 10 randomly chosen 2x2-mm fields, as described previously.¹⁸

Effect of TNF- on Protein Synthesis and Degradation in Cardiac Myocytes
Protein synthesis
The methods for determining protein synthesis in isolated adult cardiac myocytes have been described in detail.¹⁹ Briefly, a 2-mL suspension of freshly isolated cardiac myocytes was plated at a final concentration of 5x10⁴ cells/mL onto laminin-coated (20 µg/mL) polystyrene Petri dishes. In preliminary control experiments, we established that 8 hours of TNF- stimulation had no effect on cardiac myocyte protein synthetic activity; accordingly, beginning on day 1 in culture, the cardiac myocytes were exposed continuously for 24 hours to either diluent or a range of concentrations of TNF- (10 to 1000 U/mL). This range of TNF- concentrations was based on previous studies from this laboratory demonstrating that the adult feline heart produces 100 to 500 U/mL TNF- after certain forms of stress.^{20 21 22} To determine whether TNF- would increase the rate of protein synthesis in isolated cardiac myocytes, the cells were pulse-labeled for 6 hours with 30 µCi/mL [³H]phenylalanine. At the conclusion of the study, the incorporation of radiolabel was stopped by three washings of the cultures with cold (4°C) HBSS containing 10 mmol/L L-phenylalanine. The extent of radiolabeling was determined as described previously,¹⁹ and the rate of protein synthesis was calculated as phenylalanine incorporation (nmol·g protein^-1·h^-1)=(phenylalanine incorporated into total cell protein [dpm/g myocyte protein]/phenylalanine specific activity of medium [dpm/mmol])xh^-1.

The specificity of TNF-–induced effects was determined in two separate control experiments. First, we used an anti–TNF- antibody (Genzyme) to neutralize the effects of TNF- exactly as we have described previously.²³ The superfusates remaining after centrifugation of the antibody–TNF- complexes were applied to cardiac myocyte cultures for 24 hours, and protein synthesis was determined as described above. Second, we examined the rate of protein synthesis in cells stimulated with conditioned medium, which was obtained from myocyte cultures that had previously been stimulated for 24 hours with either TNF- (200 U/mL) or diluent alone. To remove any TNF- that might have remained in the conditioned medium, the medium was treated with a neutralizing anti–TNF- antibody before stimulation of the myocyte cultures.²³

Protein degradation
Beginning on day 1 in culture, the cardiac myocytes were labeled continuously for 24 hours with 30 µCi/mL [³H]phenylalanine. On day 2, the myocyte cultures were washed three times with cold HBSS containing 10 mmol/L L-phenylalanine and then exposed to medium M199 supplemented with 10 mmol/L L-phenylalanine. The cultures were then stimulated with diluent or TNF- (200 U/mL), and the amount of [³H]phenylalanine released into the culture medium at 8, 12, 24, or 36 hours was taken as a measure of protein turnover. To account for differences in release of radiolabel that might arise from differences in cell density alone, values for protein degradation were expressed as cpm/µg myocyte protein.

Effect of TNF- on Synthesis of Sarcomeric Proteins in Cardiac Myocytes
The synthesis of two sarcomeric proteins, actin and myosin heavy chain, were determined in diluent- and TNF-–stimulated (200 U/mL) myocyte cultures by previously described methods.¹⁹ Briefly, diluent- and TNF-–stimulated (200 U/mL) cultures were continuously radiolabeled for 72 hours with 30 µCi/mL [³H]phenylalanine in either the presence or absence of TNF- (200 U/mL). For these latter studies, medium was changed on days 1, 2, and 3 of culture, at which time the cell cultures were exposed to fresh medium M199 containing 30 µCi/mL of [³H]phenylalanine and fresh TNF- (200 U/mL) or diluent. After radiolabeling, the myocyte cultures were then washed with HBSS and solubilized with a buffered SDS sample buffer (4% SDS, 2% glycerol, and 0.125 mol/L Tris-HCl, pH 6.8). The diluent-stimulated (20 µg/lane) and TNF-–stimulated (20 µg/lane) samples were electrophoretically separated (7.5% SDS-PAGE) along with known molecular weight standards as well as samples of purified actin (5 µg) and myosin (5 µg). The resultant protein bands were aligned with the comigrating bands of purified actin and myosin heavy chain, excised from the gel, electroeluted, and precipitated and processed exactly as described previously.¹⁹ Results were expressed in terms of the net actin and myosin heavy chain protein synthesized. In addition to examining specific sarcomeric protein synthesis, we also examined net protein synthesis in the above cultures according to the following formula: net phenylalanine incorporation (mmol/g protein)=phenylalanine incorporated into total cell protein (dpm/g myocyte protein)/phenylalanine specific activity of medium (dpm/mmol).

Effect of Cell-Substrate Adherence on Protein Synthesis in Cardiac Myocytes
Given the importance of cell-substrate adherence for growth of adult cardiac myocytes,²⁴ we asked whether cell-substrate interaction might be important in terms of mediating the effect(s) of TNF- on net protein synthesis. Accordingly, we examined the effects of TNF- in adherent and nonadherent cardiac myocytes. Myocyte cultures were prepared and treated identically to those described above for adherent myocytes, with the exception that the cells were placed in polystyrene Petri dishes that had not been coated with laminin. Nonadherent myocyte cultures were adjusted to a final concentration of 5x10⁴ rod cells/mL and treated for 24 hours with diluent or TNF- (200 U/mL). After 24 hours of stimulation, the concentrations of rod-shaped cells in control and TNF- cultures were readjusted to 1x10⁴ cells/mL, and the cells were transferred into Petri dishes containing fresh medium M199 supplemented with 0.4 mmol/L L-phenylalanine. The cell cultures were then allowed to equilibrate for 1 hour before being pulse-labeled with 30 µCi [³H]phenylalanine. The positive control for these experiments consisted of nonadherent myocytes treated for 6 hours with 50 ng/mL phorbol 12-myristate 13-acetate dissolved in 0.01% dimethyl sulfoxide. After each set of cultures was pulse-labeled for 6 hours, the cells were transferred to conical tubes and washed and centrifuged three times with cold HBSS supplemented with 10 mmol/L L-phenylalanine. The rate of protein synthesis for myocytes was determined as described above. To determine whether TNF- exerted a toxic effect in nonadherent cardiac myocytes, we examined LDH release in the supernatants of myocyte cultures that had been treated with diluent or with TNF- (200 U/mL) for 24 hours. To account for variations in LDH release secondary to differences in cell number, the extent of LDH release was normalized by micrograms of myocyte protein (BCA assay, Pierce) per dish.

Statistical Analysis
Each value is expressed as mean±SEM. One-way ANOVA was used to evaluate mean differences in the rate of protein synthesis and net [³H]phenylalanine incorporation. Two-way ANOVA was used to evaluate differences between and within groups in protein degradation. When appropriate, post hoc multiple comparison testing was performed to test for differences between control and experimental groups (Dunnett's test) or to compare differences between experimental groups (Newman-Keuls test). Significant differences were said to exist at P<.05.

	Results

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Cardiac Myocyte Evaluation
Evaluation of the Culture System
The Table summarizes the results of the studies in which we examined the number of viable rod-shaped cardiac myocytes that remained adherent to the laminin-coated substrate. As shown, for both diluent- and TNF-–treated cultures, there was a small decrease in the absolute number of rod-shaped cells as a function of time in culture (P<.005); however, the decrease in the absolute numbers of rod-shaped cells was not significantly different (P=.78) between diluent- and TNF-–treated cultures. Further, virtually all (>98.8%) rod-shaped cells in the diluent-stimulated (n=10 dishes) and TNF-–stimulated (n=10 dishes) cultures excluded trypan blue. Thus, under the culture conditions studied here, TNF- had no obvious discernable effect on myocyte viability.

View this table: [in this window] [in a new window]   Table 1. Effect of TNF- on Cell Viability

Effect of TNF- on Protein Synthesis and Degradation in Cardiac Myocytes
Protein synthesis
The important finding shown by Fig 1 is that stimulation with TNF- led to an accelerated rate of general protein synthesis in isolated adult cardiac myocytes. As shown, treatment with 10 U/mL TNF- had no significant effect on protein synthesis, whereas stimulation with 50 U/mL TNF- resulted in a significant increase in the rate of general protein synthesis in adult cardiac myocytes. One-way ANOVA indicated that the overall changes in protein synthesis were statistically significant (P<.02); post hoc multiple comparison testing (Dunnett's) showed that there were significant differences (P<.05) from control values (diluent) for concentrations of TNF- 50 U/mL. The specificity of effects of TNF- on protein synthesis was suggested by the finding that an anti–TNF- antibody completely abolished the effects of TNF- on protein synthesis (P=.66 compared with values obtained from cultures treated with antibody alone). In addition, TNF-–conditioned medium had no significant effect (P=.61) on the rate of protein synthesis compared with values obtained for myocyte cultures treated with diluent-conditioned medium, suggesting that the stimulatory effects of this cytokine were not secondary to the secretion of a soluble growth factor into the culture medium, which then affected cell growth in an autocrine fashion.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of TNF- on rates of general protein synthesis in adherent myocytes. Freshly isolated cardiac myocytes were exposed continuously for 24 hours either to diluent or to a range of concentrations of TNF- (10 to 1000 U/mL; n=10 cultures/concentration). Beginning on day 2 in culture, 0.4 mmol/L unlabeled L-phenylalanine (PHE) was added to culture medium to ensure equalized specific activities of intracellular and extracellular phenylalanine pools43 ; the cells were then pulse-labeled for 6 hours with 30 µCi/mL [3H]phenylalanine and the cardiac myocyte proteins processed as described in "Methods." Specificity of TNF-–induced effects was determined in two separate control experiments: first, an anti–TNF- antibody (Ab) was used to neutralize effects of TNF- (left hatched bar; n=7 dishes); second, conditioned medium from TNF-– and diluent-treated cultures was applied to freshly isolated cardiac myocytes (right hatched bar; n=5 dishes) for 24 hours (see "Methods" for details). Data are depicted as ratio of rate of protein synthesis in TNF-–stimulated cells compared with diluent-treated cells to facilitate comparison between myocyte isolations. Rate (mean±SEM) of protein synthesis for control cells in these experiments was 222.8±26.4 nmol phenylalanine·g myocyte protein-1·6 h-1 (*P<.05 vs control values), similar to absolute values that have been reported in isolated feline cardiac myocytes.44

Protein degradation
Fig 2 shows that after 36 hours of stimulation with diluent or TNF-, the release of incorporated [³H]phenylalanine was relatively less for the TNF-–treated myocytes, suggesting that cytokine stimulation led to a time-dependent decrease in protein turnover. Two-way ANOVA indicated that there were both significant time-dependent (P<.0001) and group-dependent (P<.03) differences in the release of radiolabel.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of TNF- on protein degradation. Cardiac myocyte cultures were labeled continuously for 24 hours with 30 µCi/mL [3H]phenylalanine. On day 2 in culture, myocyte cultures were washed with cold 10 mmol/L L-phenylalanine and then exposed to culture medium supplemented with 10 mmol/L L-phenylalanine. Cultures were then stimulated with diluent (open bars; n=12 dishes per time point) or 200 U/mL TNF- (solid bars; n=12 dishes per time point), and amounts of [3H]phenylalanine released into culture medium were determined at specified time points. Data are depicted as the fold of change in release of [3H]phenylalanine into medium from respective baseline values obtained at 8 hours in diluent-stimulated (588.5±28.8 cpm/µg cell protein) and TNF-–stimulated (596±22.5 cpm/µg protein) cultures.

Effect of TNF- on Synthesis of Sarcomeric Proteins in Cardiac Myocytes
Fig 3 shows the results of the studies in which we examined the extent of radiolabeling of actin and myosin heavy chain proteins in diluent- and TNF-–stimulated (200 U/mL) cultures. The salient finding shown by this figure is that stimulation of the myocyte cultures with TNF- for 72 hours led to a significant 2.4-fold increase (P=.01) in net actin protein synthesis and a significant 3.3-fold increase in net myosin heavy chain synthesis (P=.02). In addition to the effects of TNF- on sarcomeric protein synthesis, we observed an 1.2-fold increase (P<.03 by ANOVA) in net incorporation of [³H]phenylalanine compared with the respective values obtained for diluent-treated cultures treated for 72 hours. Thus, these data suggest that in addition to increasing net general protein synthesis, stimulation with TNF- increases the net synthesis of specific sarcomeric proteins as well.

View larger version (26K): [in this window] [in a new window]   Figure 3. Effect of TNF- on net synthesis of sarcomeric proteins in adherent cells. TNF-–stimulated (200 U/mL) and diluent-stimulated myocyte cultures were radiolabeled for 72 hours and prepared as described in "Methods." A, Electrophoretic separation of molecular weight standards (lane 1), myosin protein (lane 2), and actin protein (lane 3) alongside comigrating actin and myosin heavy chain bands in proteins from myocytes treated with diluent (lane 4) or TNF- (lane 5). The actin (42 kD) and myosin heavy chain (205 kD) bands were aligned with respective protein standards and excised from gel (see "Methods" for details); proteins were electroeluted from gel pieces, and samples were split for protein determination and scintillation counting. B, Results of group data for net synthesis of new actin (n=10 culture dishes for TNF- and diluent control) and myosin heavy chain protein (n=10 culture dishes for TNF- and diluent control) in diluent- and TNF-–stimulated cultures. *P<.01; **P<.02. PHE indicates phenylalanine.

Effect of Cell-Substrate Adherence on Protein Synthesis
In contrast to the effects of TNF- on adherent cardiac myocytes, treatment of nonadherent myocyte cultures with TNF- (200 U/mL) had no significant effect (P=.68) on the rate of protein synthesis (373.5±36 nmol phenylalanine·g myocyte protein^-1·6 h^-1; n=10 dishes) compared with diluent-treated control cells (355.7±22.8 nmol phenylalanine·g myocyte protein^-1·6 h^-1; n=10 dishes), despite use of a concentration of TNF- (200 U/mL) that was maximally stimulatory in cultures of adherent cardiac myocytes. To be certain that the nonadherent cells were competent to increase their rate of protein synthesis, we treated cultures of nonadherent cardiac myocytes with a phorbol ester. This study showed that there was a significant 1.3-fold increase (P<.01 compared with control) in the rate of protein synthesis in the nonadherent cells treated with phorbol ester (448.1±26 nmol phenylalanine·g myocyte protein^-1·6 h^-1; n=10 culture dishes) compared with diluent-treated control cells. Finally, to exclude the possibility that TNF- exerted a toxic effect in nonadherent cardiac myocytes, we examined LDH release in myocyte cultures that had been treated with diluent or with TNF- (200 U/mL). This study showed that there was no significant difference (P=.97) in the extent of LDH release in diluent-treated cells (1.25±0.35 U/µg cell protein, n=6 dishes) compared with TNF-–treated cultures (1.27±0.46 U/µg cell protein, n=6 dishes). Taken together, these results suggest that cell-substrate adherence is important for the growth-stimulatory effects of TNF- in cardiac myocytes.

	Discussion

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The major conclusion to be drawn from this study is that physiologically obtainable concentrations of TNF- provoke a hypertrophic growth response in the adult mammalian cardiac myocyte. The following three lines of evidence support this statement. First, when adult cardiac myocytes were stimulated with TNF-, there was an increase in the rate of general protein synthesis (Fig 1) for concentrations of TNF- 50 U/mL. It bears emphasis that the concentrations of TNF- that were necessary to stimulate protein synthesis in cardiac myocytes are available to the cardiac myocyte during certain forms of stress^{20 21 22} and do not necessarily produce negative inotropic effects in isolated contracting cardiac myocytes.²³ Moreover, the effects of TNF- on net protein synthesis were shown to be dependent on cell-substrate interaction, which suggests (but does not prove) that the cell signaling pathways used by TNF- are dependent on a preserved interaction between cell integrins and the extracellular matrix. The specificity of the TNF-–induced effects was demonstrated both by the studies in which a neutralizing anti–TNF- antibody blocked the stimulatory effects of TNF- (Fig 1) and by the studies in which TNF-–conditioned medium failed to stimulate increased protein synthesis in isolated cardiac myocytes (Fig 1). Second, stimulation of the myocyte cultures with TNF- resulted in a significant time-dependent decrease in protein degradation compared with diluent-treated control cultures (Fig 2). Third, TNF- stimulation produced significant 2.4- and 3.3-fold increases, respectively, in the synthesis of two sarcomeric proteins: actin and myosin heavy chain protein (Fig 3).

TNF- as an Autocrine/Paracrine Mediator of Myocardial Remodeling
TNF- is a pleiotropic cytokine whose net biological effect may ultimately be beneficial or, alternatively, detrimental for the host, depending on the quantity released and the specific microenvironment in which this cytokine exerts its effects. Pertinent to this discussion is the observation that the adult mammalian cardiac myocyte synthesizes TNF- mRNA and protein de novo after certain forms of stress.^{6 7 8} Although the precise biological role for stress-induced TNF- expression within the heart is unknown, two lines of evidence suggest that the expression of TNF- may subserve an important autocrine and/or paracrine role in modulating myocardial homeostasis. First, TNF- gene expression is not constitutive within the heart but rather is temporally coupled to stressful environmental stimuli.^{6 7 8} TNF- biosynthesis within the heart is not only extremely rapid in onset, occurring within 60 minutes after the onset of a stressful stimulus, but also rapid in offset, insofar as TNF- mRNA levels return to baseline within 90 minutes after the removal of the inciting stress.⁶ Second, stimulation with TNF- provokes increased expression of 27- and 72-kD heat shock proteins in fetal and adult cardiac myocytes, respectively.^{25 26} Given that members of the 70-kD family of heat shock proteins are thought to promote tissue repair in the heart,^{27 28} TNF- stimulation may confer protective resistance to environmental stress in the cardiac myocyte through upregulation of protective stress proteins. Third, TNF- is known to activate a family of matrix metalloproteinases, including collagenase type 1, stromelysin-1, and gelatinase A and B,^{29 30 31 32} which are capable of degrading the components of the extracellular matrix and thereby promoting remodeling of the ventricular chamber in response to a superimposed environmental stress. The observation in the present study that TNF- provokes a modest hypertrophic growth in cardiac myocytes provides a fourth line of evidence that suggests that TNF- may subserve an important role in modulating myocardial homeostasis. That is, stress-induced elaboration of TNF- might stimulate increased synthesis of sarcomeric and stress proteins in cardiac myocytes in an autocrine/paracrine manner and thus provide the myocyte with a short-term adaptive response to stress. Although the above statements remain speculative for the present, they are entirely consistent with recent important studies in cultured neonatal cardiac myocytes suggesting that proinflammatory cytokines such as IL-1ß and TNF- are sufficient to stimulate hypertrophic growth responses in neonatal cardiac myocytes^{3 15 33} and are consonant with a recent report in which the overexpression of IL-6 and IL-6 receptor transgenes in mice led to the development of cardiac hypertrophy.³⁴ Given that TNF- can induce the expression of both IL-1 and IL-6,^{35 36} one may postulate that the myocardial response to environmental stress involves activation of a hierarchical cytokine cascade that is initiated by TNF- and is then sustained by sequential activation of a network of downstream cytokines, including IL-1ß and IL-6, which act in concert with TNF- to facilitate repair and remodeling in the heart.

Conclusions
The precise homeostatic mechanisms that are elicited by the heart in response to stressful environmental stimuli, such as superimposed hemodynamic overloading or myocardial infarction, remain poorly understood. Nevertheless, there is a growing appreciation that the myocardial adaptation to stress and/or injury is far from simple and probably involves specific biochemical responses by myocardial cells and nonmyocardial cells that reside in the heart as well as specific alterations in the vast array of extracellular matrix proteins that support these "cardiocytes." One intriguing question that arises from the above discussion is, how does the heart integrate and coordinate the various intracellular and extracellular components of the environmental response to stress?

One of the primary roles that cytokines subserve in any biological system is that of enabling cells to communicate via juxtacrine, paracrine, or autocrine pathways. Although the precise role for the expression of TNF- within the heart remains unknown at present, given the rapidity with which TNF- mRNA and protein are expressed by cardiac myocytes after environmental stress^{20 21} and given that TNF- is sufficient to modulate growth in myocardial and nonmyocardial cells in the heart and to protect cells from ischemia-reperfusion injury through upregulation of manganese superoxide dismutase^{37 38} as well as to remodel the extracellular matrix,^{29 39 40} it is becoming increasingly likely that the elaboration of TNF- within the heart may serve as an important regional mechanism for coordinating and integrating critical homeostatic responses to stress in the heart. Nonetheless, it should be recognized that the above statements are confined to the short-term effects of physiological concentrations of TNF- within the heart and that the sustained long-term overexpression of TNF- may be frankly maladaptive,⁴¹ perhaps through the induction of cardiac myocyte apoptosis.⁴² Accordingly, it will be increasingly important in future studies to determine the basic mechanisms that regulate physiological and pathophysiological expression of TNF- within the heart.

	Selected Abbreviations and Acronyms

 

HBSS = Hanks' balanced salt solution HSA = human serum albumin IL = interleukin LDH = lactic dehydrogenase TNF- = tumor necrosis factor-

	Acknowledgments

 
This research was supported by research funds from the Department of Veterans Affairs and funds from the NIH (K08HL-02010, P50-HL-06H, and HL-42550-6/10). The authors gratefully acknowledge the technical assistance of Dorellyn Lee-Jackson and the secretarial assistance of Jana Grana. The authors are also indebted to Dr Andrew I. Schafer for his past and present support.

	Footnotes

 
Reprint requests to Douglas L. Mann, MD, Cardiology Section (111B), VA Medical Center, 2002 Holcombe Blvd, Houston, TX 77030. E-mail dmann@bcm.tmc.edu.

Guest editor for this article is Judith L. Swain, Stanford University Medical Center, Palo Alto, Calif.

Received January 29, 1996; revision received August 26, 1996; accepted October 23, 1996.

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