Tumor Necrosis Factor-α Mediates Cardiac Remodeling and Ventricular Dysfunction After Pressure Overload State
Background— Pressure overload is accompanied by cardiac myocyte apoptosis, hypertrophy, and inflammatory/fibrogenic responses that lead to ventricular remodeling and heart failure. Despite incomplete understanding of how this process is regulated, the upregulation of tumor necrosis factor (TNF)-α after aortic banding in the myocardium is known. In the present study, we tested our hypothesis that TNF-α regulates the cardiac inflammatory response, extracellular matrix homeostasis, and ventricular hypertrophy in response to mechanical overload and contributes to ventricular dysfunction.
Methods and Results— C57/BL wild-type mice and TNF-knockout (TNF−/−) mice underwent descending aortic banding or sham operation. Compared with sham-operated mice, wild-type mice with aortic banding showed a significant increase in cardiac TNF-α levels, which coincided with myocyte apoptosis, inflammatory response, and cardiac hypertrophy in week 2 and a significant elevation in matrix metalloproteinase-9 activity and impaired cardiac function in weeks 2 and 6. Compared with wild-type mice with aortic banding, TNF−/− mice with aortic banding showed attenuated cardiac apoptosis, hypertrophy, inflammatory response, and reparative fibrosis. These mice also showed reduced cardiac matrix metalloproteinase-9 activity and improved cardiac function.
Conclusions— Findings from the present study have suggested that TNF-α contributes to adverse left ventricular remodeling during pressure overload through regulation of cardiac repair and remodeling, leading to ventricular dysfunction.
Received June 2, 2006; accepted November 17, 2006.
Congestive heart failure has emerged as a major health problem during the past 2 decades. Myocardial remodeling has been demonstrated to contribute significantly to ventricular dysfunction in various heart diseases, including myocardial infarction, cardiomyopathy, and hypertensive heart disease. Pressure overload is accompanied by various cardiac molecular and cellular changes, including myocyte apoptosis, inflammatory/fibrogenic responses, and hypertrophy, which lead to myocardial remodeling and subsequent ventricular dysfunction.1–3 The factors that regulate cardiac events during pressure overload, however, are continuing to evolve.
Clinical Perspective p 1407
Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine elaborated by all cardiac cells after stress to stimulate an inflammatory response.4,5 TNF-α also triggers intracellular signaling cascades that modulate host defense against injury, facilitate growth and survival, and promote apoptosis and matrix metalloproteinase (MMP) expression.6–8 Circulating and cardiac TNF-α levels are elevated in heart diseases such as dilated cardiomyopathy, myocardial infarction, and left ventricular (LV) pressure overload.9,10 Consequently, TNF-α has been implicated in the pathogenesis of ventricular remodeling in the infarcted heart and in cardiac dysfunction.4,8,11,12
Cardiac TNF-α expression is upregulated with pressure overload. Its importance in cardiac remodeling and dysfunction after pressure overload state has not been fully elucidated, however. In the present study, we sought to determine whether TNF-α is involved in cardiac molecular and cellular changes during pressure overload and associated with heart failure. Using aortic banding in wild-type (WT) mice and TNF-α gene knockout mice, we detected the potential regulation of TNF-α in cardiac inflammatory/fibrogenic responses, apoptosis, hypertrophy, and ventricular function during pressure overload.
Cardiac myocytes were isolated from the ventricles of 1-day-old neonatal mice. Myocytes were dissociated by 0.15% trypsin in 137 mmol/L NaCl, 5.369 mmol/L KCl, 0.81 mmol/L MgSO4, 5.55 mmol/L dextrose, 0.44 mmol/L KH2PO4, 0.34 mmol/L Na2HPO4.7H2O, and 20.06 mmol/L Hepes (pH 7.4). Cells were differentially plated for 1 hour to remove fibroblasts and other contaminating cell types. Then, cells were seeded onto flexible-bottom 6-well laminin-coated culture plates (Flecell, Hillsborough, NC) at a density of 2×105 cells per well and cultured initially with DMEM/F12 medium supplemented with 10% fetal bovine serum for 24 hours and then rested after 24 hours of serum-free condition. Myocytes were randomized to a stretch stimulus of 25% elongation at 30 cycles per minute for 0, 10, 30, and 60 minutes and harvested. RNA was extracted by TRIzol (GIBCO, Invitrogen, Carlsbad, Calif) and subjected to reverse-transcriptase polymerase chain reaction to determine the expression of TNF-α as previously reported.13
Descending aortic banding was performed in 12-week-old male (25 to 27g) WT littermates and TNF−/− mice (C57BL/6-TNFtm1Gk1, Jackson Laboratory, Bar Harbor, Me). Mice were anesthetized; a horizontal skin incision was made at the level of the 2 to 3 intercostals space; the descending aorta was isolated; and a 7-0 silk suture was snared with wire and pulled back around the aorta. A bent 27-gauge needle was then placed next to the aorta, and the suture was tied snugly around the needle and the aorta. After ligation, the needle was quickly removed, the chest and skin were closed, and the mice were allowed to recover. To serve as controls, age-matched animals underwent the same procedure without placement of the aortic banding.
Animals were euthanized on postoperative weeks 2 and 6 (n=8 surviving animals per time point per group). Hearts were harvested, rinsed with cold PBS, and frozen and stored at −80°C for immunohistochemical, biochemical, and histochemical studies.
On postoperative week 6, cardiac function was detected (n=12 surviving animals per time point per group). After the hemodynamic measurements, hearts were perfusion-fixed and sectioned for cardiac morphometrical study.8
Protein Cytokine Antibody Array
Tissue samples (n=4) were analyzed with cytokine antibody array by using RayBio Mice Cytokine Antibody Array I (RayBiotech, Inc, Norcross, Ga) according to the manufacturer’s instructions. Briefly, cytokine array membranes were blocked in 2 mL blocking buffer for 30 minutes and then incubated with 1 mL sample (500 μg protein) at 4°C overnight. Samples were then decanted from each container, and membranes were washed 3 times with 2 mL wash buffer 1, followed by 2 washes with 2 mL of washing buffer 2 at room temperature. Membranes were then incubated in 1:250-diluted biotin-conjugated primary antibodies at room temperature for 2 hours, washed with washing buffer, and incubated in 1:1000-diluted horseradish peroxidase–conjugated streptavidin for 1 hour. Membranes were washed thoroughly, exposed to a peroxidase substrate for 5 minutes in the dark, and exposed on x-ray film (Kodak X-OMAT AR film, Kodak, Rochester, NY) for 30 minutes. A semiquantitative analysis of the comparative intensity of the spots was performed with image analysis system (NIH Image 1.60, National Institutes of Health, Bethesda, Md).
Cryostat sections (5 μm) were prepared, air-dried, and fixed in 4% paraformaldehyde/PBS for 15 minutes. The sections were incubated with 0.3% hydrogen peroxide and 10% BSA for 15 minutes. The sections were then incubated with antibody against TNF-α and CD45 (BD PharMingen Technical, Mississauga, Ontario, Canada) and atrial natriuretic factor (ANF) at 4°C overnight. The sections were then incubated with a matching biotinylated secondary antibody (Vector, Burlingame, Calif) for 45 minutes at room temperature. Negative controls were performed for all immunological staining by omission of the primary antibody. Similar procedures were followed for inflammatory cell immunotyping, using antibodies to CD3 (BD PharMingen Technical) to detect lymphocytes and Mac-3 to detect macrophages.
Western Blot Analysis
Cardiac tissue was homogenized with lysis buffer and diluted 1:1 with 2× SDS sample buffer (Invitrogen Novex, Burlington, Ontario). An equal amount of protein (20 μg) was loaded onto each lane of 8% to 16% Tris-Glycin gel (Helixx, Toronto, Ontario, Canada). Proteins were then separated by electrophoresis and transferred from the gel to a nitrocellulose membrane with an electroblotting apparatus. Membranes were incubated with 5% BSA for 1 hour to decrease nonspecific sites. Samples were then incubated with antibody against transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), ANF, or caspase-8 (Santa Cruz Biochemistry Inc, Santa Cruz, Calif) overnight at 4°C. Samples were then washed and subsequently incubated with peroxidase-conjugated secondary antibody and detected with the ECL Detection Kit (Amersham, Piscataway, NJ).
Cardiac protein was extracted with lysis buffer after homogenization. Samples (20 μg) were loaded onto each lane of 10% zymogram gelatin minigels (Novex, San Diego, Calif). Gels were run at 35 mA for 2.5 hours and then incubated twice in 2.5% Triton-X 100 for 1 hour at room temperature, washed, and incubated for another 16 hours in 50 mmol Tris-HCl buffer, pH 7.5, containing 200 mmol NaCl and 10 mmol CaCl2 at 37°C. Gels were stained for 90 minutes in Coomassie blue and destained in 30% methanol/10% acetic acid for 60 minutes. White bands on a blue background indicated zones of digestion corresponding to the presence of different MMPs identified from their molecular weight. Bands were scanned with a densitometer (GS-700 Bio-Rad, Hercules, Calif). MMP levels were quantified with the Multianalyst software (Bio-Rad).
Detection of DNA Fragmentation
The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was used to monitor the extent of DNA fragmentation as a measure of apoptosis in cryostat sections as previously reported.8
Cardiac Collagen Volume Fraction
Cardiac collagen volume fraction was determined on picrosirius red–stained sections. The cardiac collagen volume fraction was calculated as the ratio of the sum of total interstitial collagen area to the sum of total collagen and noncollagen area in the entire visual field of the section as previously reported.8
Morphometric analysis was performed on cardiac sections with an image quantitative digital analysis system (NIH Image 1.6). Relative ventricular cavity and wall thickness were determined according to the method of Sun et al.8 A single myocyte was measured with images captured from hematoxylin and eosin–stained sections. The outline of 100 to 200 myocytes was traced in each section. Simple polymerase chain reaction image system software (Universal Imaging, Media, Pa) was used to determine myocyte cross-sectional area.
At 6 weeks after aortic banding, mice were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) and placed on controlled heating pads. Core temperature was measured with a rectal probe and maintained at 37±0.5°C. A microtip catheter transducer (SPR-839, Millar Instruments, Houston, Tex) was inserted into the right carotid artery and advanced into the left ventricle under pressure control. After stabilization for 15 minutes, the pressure signals were recorded continuously with an ARIA pressure-volume conductance system coupled with a Powerlab/4SP A/D converter, stored, and displayed on a personal computer. PVAN software (Millar Instruments) was used for subsequent analysis of pressure-volume loops.
Statistical analyses of parameters of LV morphometric features, apoptosis, cardiac function, MMP activity, and collagen volume fraction were performed with ANOVA among animal groups at week 2 or 6. Multiple subgroup comparisons were made by Scheffé’s F test. Values were expressed as mean±SEM, with values of P<0.05 considered significant.
All authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
TNF and In Vitro Myocyte Stretch Model
In an initial in vitro study, mechanical stretching of neonatal myocytes induced a surprisingly rapid expression of TNF-α mRNA in cardiomyocytes (Figure 1A). To further investigate the effect of mechanical stimulation on local TNF-α expression and subsequent LV remodeling on the basis of this finding, we proceeded with an in vivo cardiac pressure overload model by aortic banding in TNF-α knockout mice and its WT littermates.
Mortality in sham-operated TNF−/− and WT mice was zero. At 6 weeks after aortic banding, mortality in WT and TNF−/− mice was 25% and 6%, respectively.
Cardiac TNF-α Expression and Inflammatory Response
Cardiac TNF-α expression was detected by immunohistochemistry. Compared with sham-operated mice, cardiac TNF-α labeling was largely increased in WT mice with aortic banding but was not detected in TNF−/− mice with aortic banding (Figure 1B). This finding was confirmed by cytokine antibody array (Figure 1C). To evaluate the inflammatory response, we detected inflammatory cell infiltration and expression of proinflammatory cytokines in the heart after aortic banding by immunohistochemical CD45 labeling and cytokine antibody array, respectively. At week 2 after aortic banding, abundant inflammatory cells appeared in the left ventricle of WT animals. Most of these inflammatory cells were lymphocytes and monocyte-derived macrophages. Very few neutrophils were observed in these regions. However, only a few inflammatory cells were seen in the hearts of TNF−/− mice with aortic banding (Figure 1B). Compared with sham-operated mice, cardiac inflammatory cytokine, interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein (MIP)-1 were significantly elevated in WT mice with aortic banding, whereas only slight increases in cardiac IL-6, MCP-1, and MIP-1 were seen in TNF−/− mice with pressure overload (Figure 1C and 1D).
Apoptotic cardiomyocytes were rarely seen in the heart of sham-operated WT and TNF−/− mice (Figure 2A and 2B). After aortic banding, however, 0.91% and 0.57% of myocytes underwent apoptosis in WT mice at weeks 2 and 6, respectively, whereas in TNF−/− mice, myocyte apoptosis rate was 0.26% at week 2 and 0.195% at week 6 after aortic banding (Figure 2A and 2B). By Western blot, cardiac caspase-8 protein levels were significantly increased in WT mice with aortic banding compared with sham-operated controls. There was no significant difference in cardiac caspase-8 level between sham-operated mice and TNF−/− mice after aortic banding (Figure 2C).
In the normal heart, a small amount of collagen is present in the interstitial and perivascular space (Figure 3A). Cardiac collagen volume was greatly increased in WT mice at week 6 after aortic banding, with a diffuse small patchy and nonuniform pattern, as well as collagen network structure destruction and disorganization in the interstitial area. Collagen volume in TNF−/− mice with aortic banding also was increased but to a lesser extent than in WT mice, which displayed uniformly increased densities (Figure 3A). The quantitative data of cardiac collagen volume are shown in Figure 3B.
TGF-β and CTGF protein are the major regulators for fibrous tissue formation. We evaluated myocardial TGF-β and CTGF protein levels by Western blot. Cardiac TGF-β and CTGF protein levels were significantly increased in WT mice with aortic banding compared with sham-operated mice (Figure 4A). Cardiac TGF-β and CTGF levels also were increased in TNF−/− mice, but this increase was less evident compared with WT mice. The quantitative data of cardiac TGF-β and CTGF levels are shown in Figure 4B.
Aortic banding resulted in the appearance of LV hypertrophy in WT mice within 2 weeks after aortic banding and became more evident at 6 weeks. In WT mice with aortic banding, the heart-to-body weight ratio increased by 69% and their LV myocyte size increased by 72% compared with sham-operated controls. In contrast, TNF deficiency partially preserved LV hypertrophy for at least 6 weeks. The heart-to-body weight ratio increased by 39% and LV myocytes enlarged by 41% (Figure 5A and 5B). Morphometry studies showed that LV wall thickness was progressively increased up to 37% at 2 weeks and become 21.6% at 6 weeks in WT mice after aortic banding. In mice lacking TNF, LV thickness increased by 13.6% and 29.3% at 2 and 6 weeks, respectively, after aortic banding (Figure 5B). Furthermore, hypertrophy also was accompanied by the induction of cardiac ANF. Labeling of hypertrophied myocytes with anti-ANF antibody (Figure 5C, a) showed increased ANF protein levels in both WT and TNF−/− mice, whereas ANF levels were significantly greater in WT mice than in TNF−/− mice (1.45±0.051 for WT versus 1.05±0.041 for TNF−/−; P<0.001) at 6 weeks after aortic banding (Figure 5C, b, c).
Cardiac MMP Expression
Detected by zymography, cardiac MMP-9 activity was significantly increased in WT mice at weeks 2 and 6 after aortic banding compared with controls. MMP-9 activity also was slightly increased at 2 weeks after aortic banding in TNF−/− mice but was much less significant than in WT mice. Cardiac MMP-2 activity was increased in both WT and TNF−/− mice compared with sham-operated controls but was not statistically significantly different between TNF−/− and WT mice (Figure 6A and 6B).
LV cardiac function was evaluated in vivo with a miniaturized 1.4F conductance catheter. End-systolic and end-diastolic pressures and volumes and ejection fraction were similar in sham-operated WT and TNF−/− mice (the Table). Our study showed that LV end-systolic and end-diastolic pressures were significantly increased in WT and TNF−/− mice 2 weeks after aortic banding (data not shown). LV end-systolic and end-diastolic volumes were elevated at 6 weeks, whereas LV end-systolic pressure, ejection fraction, and ±dP/dt were significantly decreased in WT mice with aortic banding. However, in TNF−/− mice with aortic banding, LV end-systolic pressure maintained significantly higher than in WT mice with aortic banding, whereas ejection fraction was not significantly different compared with controls (the Table).
Pressure overload is accompanied by a variety of cardiac cellular and molecular changes that lead to myocardial remodeling and subsequent ventricular dysfunction.14,15 There is growing recognition of and experimental evidence to support the regulatory role of TNF-α in the pathogenesis of myocardial repair/remodeling in various cardiac diseases. In the present study, we addressed the potential contribution of TNF-α to cardiac molecular/cellular events and ventricular dysfunction during pressure overload.
The present study demonstrates that cardiac levels of TNF-α, which is not constitutively expressed in the normal heart, are significantly increased in WT mice after aortic banding. Furthermore, this increase in cardiac TNF-α level after aortic banding is coincident with the upregulation of IL-6, MCP-1, MIP-1γ, and inflammatory cell accumulation in the left ventricle of WT mice. These findings suggest that TNF-α is involved in cardiac inflammatory response in animals with pressure overload. In contrast, cardiac cytokine production and inflammatory response are less evident in TNF−/− mice with aortic banding, which supports the contribution of TNF-α to pressure overload–induced cardiac inflammatory response. It is known that TNF-α can promote the inflammatory response by stimulating inflammatory protein synthesis, macrophage phagocytosis, and cell growth and differentiation. Our study also demonstrates that TNF-α–producing cells in the heart include inflammatory cells and myocytes in WT mice with aortic banding. In vivo and in vitro studies have demonstrated that cardiac myocytes produce substantial amounts of TNF-α in response to mechanical stress.4,16 Cardiac TNF-α expression also is upregulated with other cardiac diseases, including myocardial infarction, in which it is involved in the cardiac inflammatory response.8 It has been demonstrated that other chronic inflammatory diseases such as rheumatoid arthritis and Crohn’s disease also are associated with increased local production of TNF. Blockage of this cytokine with bioengineered compounds has significantly changed the therapy of these diseases and has ushered in the era of biological therapy.17,18 Clinically, immunosuppressive therapies have demonstrated the ability to improve symptoms and LV ejection in selected patients with dilated cardiomyopathy and clear signs of myocardium inflammation. Despite these interesting results in preliminary clinical studies, large clinical trials have not proved the effectiveness of the biological agents that bind TNF in the circulation of heart failure patients.19 This raises the possibility that either the pathophysiological hypotheses were not correct or the biological agents used to block TNF were not appropriate.
Loss of cardiomyocytes is an important mechanism in the development of myocardial remodeling and cardiac failure.20 After aortic banding, myocyte apoptosis was largely increased in the left ventricle of WT mice. However, myocyte apoptosis was rarely seen in TNF−/− mice with pressure overload. TNF-α has been proved to stimulate cardiomyocyte apoptosis in various cardiac diseases. The present study has confirmed previous findings. TNF-α may trigger cardiomyocyte apoptosis via more than 1 mechanism. We observed elevated cardiac caspase-8 levels in WT mice with aortic banding, which indicates the activation of a caspase-dependent apoptotic pathway in response to TNF signaling. TNF-α–induced myocyte apoptosis also can be mediated by oxidative stress,21,22 which can be inhibited by antioxidants such as thioredoxin and N-acetylcysteine.23
Cardiac fibrosis is a feature of cardiac remodeling after aortic banding. We observed reparative fibrosis (focal fibrosis), which replaces damaged myocytes in WT mice at 6 weeks after aortic banding, whereas in TNF−/− mice, there was a trend toward a lesser or absent amount of locally focal fibrosis compared with WT mice. These findings suggest that TNF-α is involved in cardiac damage/reparative fibrosis. Enhanced TGF-β and CTGF expression in mice with aortic banding was observed in WT mice. CTGF is well known for its role as a downstream mediator of the chronic fibrotic effects of TGF-β. Activated by TGF-β, CTGF stimulates fibroblasts to differentiate into myofibroblasts, the key cells for collagen synthesis in the repaired heart.24–26
WT and TNF−/− mice with aortic banding developed various degrees of cardiac hypertrophy. LV hypertrophy is an initially expected response of the heart to pressure overload, which permits the cardiomyocytes to generate additional force to overcome the increase in pressure load. Unfortunately, the initial response may become maladaptive when cardiomyocytes degenerate as a result of ischemia, inflammatory response, fibrosis, and progressive LV dilation with a progressive decline in cardiac pump function.27–31 Our studies show that at 6 weeks after aortic banding, WT mice underwent a transition from compensated hypertrophy to heart failure characterized by depressed LV contractility. Compared with WT mice, in TNF−/− mice at 6 weeks after aortic banding, cardiac function is partially preserved, suggesting that the appearance of cardiac dysfunction induced by pressure overload may be protected or delayed in TNF−/− mice. These observations are consistent with previous studies that showed the development of LV dysfunction and dilated cardiomyopathy in transgenic mice overexpressing TNF-α.32
Generally, cardiac function requires the orchestration of multiple mechanical and biological factors within the myocardial microenvironment. The cellular matrix provides a structural foundation for the myocytes, fibroblasts, and endothelial cells to build an architectural network and provides an environment for cell signaling, cell–cell interactions, etc. In the present study, LV dysfunction was observed in WT mice at 6 weeks after aortic banding. Enhanced collagen degradation has been shown to contribute to ventricular dysfunction. MMPs, which degrade most of the extracellular matrix components, are normally present in the myocardial interstitium in an inactive form and can be activated by cytokines and counterregulated to a certain extent by tissue inhibitors of MMPs.33 In the injured heart, MMP-9 is secreted primarily by inflammatory cells, as well as by myocytes, fibroblasts, and smooth muscle cells.34,35 Our study showed markedly increased cardiac MMP-9 levels in WT mice with pressure overload but not in TNF−/− mice, which suggests that TNF-α triggers cardiac MMP-9 expression and activation with pressure overload. Our finding supports the previous finding of Kubota et al.36 Increased MMP expression may be responsible for the extracellular matrix dysregulation during pressure overload resulting from alterations in the amount, type, stability, and organization of fibrillar collagen.
In summary, by using WT and TNF−/− mice with aortic banding, we studied the potential regulation of TNF-α on cardiac remodeling and ventricular dysfunction. Our study has demonstrated that locally generated TNF is involved in cardiac remodeling by stimulating apoptosis, inflammatory and fibrogenic responses, myocyte hypertrophy, and MMP activation in pressure-overloaded heart, which, in turn, contribute to cardiac dysfunction.
Dr Liu is the Heart & Stroke/Polo Chair Professor of Medicine and Physiology, University of Toronto, and scientific director, Institute of Circulatory and Respiratory Health, Canadian Institutes of Health Research. Dr Kirshenbaum is a Canada Research Chair in Molecular Cardiology at University of Manitoba.
Sources of Funding
The present study was supported in part by grants from the Heart and Stroke Foundation of Ontario, the Canadian Institutes of Health Research (CIHR), and the Team Research Program (CHENET) and Group Program (CHF-CORE) from CIHR.
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Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 1997; 385: 729–733.
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Kapadia SR, Oral H, Lee J, Nakano M, Taffet GE, Mann DL. Hemodynamic regulation of tumor necrosis factor-alpha gene and protein expression in adult feline myocardium. Circ Res. 1997; 81: 187–195.
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Persistent pressure overload stimulates ventricular hypertrophy. In certain individuals, this can lead to ventricular remodeling, ventricular dysfunction, and heart failure. Many growth signals have been identified that can mediate this hypertrophy response. However, the factors that may trigger the subsequent transition to adverse remodeling and ventricular dysfunction are not as clear. In the present study, we demonstrate in a murine model that the heart can generate a significant amount of stress-induced proinflammatory hormones such as tumor necrosis factor directly in the myocytes during pressure overload. Excessive tumor necrosis factor, however, is associated with myocyte death, tissue fibrosis, ventricular remodeling, and cardiac dysfunction. Conversely, when tumor necrosis factor is genetically deleted from the mouse, despite the same pressure overload, there is marked improvement in ventricular function and a reduction in adverse remodeling compared with the normal animals. This is accompanied by amelioration of myocyte death and fibrosis and reduced levels of matrix modulating enzyme such as matrix metalloproteinase-9. This finding suggests that local cardiac activation of factors such as tumor necrosis factor and matrix metalloproteinase-9 may mediate the important transition to ventricular dysfunction and failure after pressure overload. This may indicate a new direction for markers of transition to heart failure and possible future strategies to prevent this progression to protect the host.