Charles F. McTiernan

Dilated Cardiomyopathy in Transgenic Mice With Cardiac-Specific Overexpression of Tumor Necrosis Factor-α

The failing human heart expresses tumor necrosis factor-alpha (TNF-alpha). However, its pathophysiological significance is not clear. We previously reported that robust overexpression of TNF-alpha in the murine heart causes lethal myocarditis. In this study, we modified the transgene to reduce the production of TNF-alpha by preserving the destabilizing sequence in TNF-alpha cDNA. Expression was driven by the murine alpha-myosin heavy chain promoter. Use of this modified construct allowed to the establish a mutine transgenic line (TG). TG offspring were examined at 6, 12, and 24 weeks. All showed a significantly higher heart weight-to-body weight ratio. Northern blot analysis confirmed the expression of transgene in the heart, and enzyme-linked immunosorbent assay demonstrated the presence of TNF-alpha protein. The TG heart demonstrated a mild, diffuse, lymphohistiocytic interstitial inflammatory infiltrate. Cardiomyocyte necrosis and apoptosis were present but not abundant. Magnetic resonance imaging showed that the TG heart was significantly dilated with reduced ejection fraction. Although the left ventricular dP/dtmax was not different at baseline, its responsiveness to isoproterenol was significantly blunted in TG. Atrial natriuretic factor was expressed in the TG ventricle. A group of TG died spontaneously, and subsequent autopsies revealed exceptional dilation of the heart, increased lung weight, and pleural effusion, suggesting that they died of congestive heart failure. The cumulative mortality rate at 6 months was 23%. In conclusion, the mouse overexpressing TNF-alpha recapitulated the phenotype of congestive heart failure. This provides a novel model to elucidate the role of this cytokine in the development of congestive heart failure.

Differential Expression of Tissue Inhibitors of Metalloproteinases in the Failing Human Heart

BACKGROUND Extracellular matrix turnover is regulated by matrix metalloproteinases (MMPs) and a family of tissue inhibitors of metalloproteinases (TIMPs). Together, these proteins may contribute to myocardial remodeling in congestive heart failure. We hypothesized that the expression of MMPs and TIMPs might be differentially regulated in the failing human heart. METHODS AND RESULTS Northern blot analyses were performed with probes to TIMP-1 to -4 and GAPDH with poly A+ mRNA from ventricular tissues of patients with ischemic cardiomyopathy (ICM, n=16) or idiopathic dilated cardiomyopathy (DCM, n=15) and nonfailing control hearts (n=15). TIMP-1 to -4 and MMP-9 proteins were quantified by ELISA and/or Western blot, and the total gelatinolytic activity was studied by gelatin zymography. The results showed that cardiac expression of TIMP-1 and -3 transcripts and proteins was significantly reduced in ICM and DCM. No significant difference was observed in TIMP-2 and -4 transcripts. However, TIMP-4 protein was significantly reduced in ICM myocardium. MMP-9 protein content and total gelatinolytic activity were upregulated in the same samples. CONCLUSIONS These studies demonstrated a selective downregulation of TIMPs along with upregulation of MMP-9 and gelatinolytic activity in the failing hearts, alterations that favor matrix degradation and turnover. These findings might be of pathophysiological significance and might suggest new therapeutic targets for limiting the ventricular remodeling and dilatation process characteristic of the failing human heart.

Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling.

Myocardial fibrosis due to maladaptive extracellular matrix remodeling contributes to dysfunction of the failing heart. Further elucidation of the mechanism by which myocardial fibrosis and dilatation can be prevented or even reversed remains of great interest as a potential means to limit myocardial remodeling and dysfunction. Matrix metalloproteinases (MMPs) are the driving force behind extracellular matrix degradation during remodeling and are increased in the failing human heart. MMPs are regulated by a variety of growth factors, cytokines, and matrix fragments such as matrikines. In the present report, we discuss the regulation of MMPs, the role of MMPs in the development of cardiac fibrosis, and the modulation of MMP activity using gene transfer and knockout technologies. We also present recent findings from our laboratory on the regulation of the extracellular MMP inducer (EMMPRIN), MMPs, and transforming growth factor-beta(1) in the failing human heart before and after left ventricular assist device support, as well as the possibility of preventing ventricular fibrosis using different anti-MMP strategies. Several studies suggest that such modulation of MMP activity can alter ventricular remodeling, myocardial dysfunction, and the progression of heart failure. It is therefore suggested that the interplay of MMPs and their regulators is important in the development of the heart failure phenotype, and myocardial fibrosis in heart failure may be modified by modulating MMP activity.

Cardiac-specific overexpression of tumor necrosis factor-alpha causes lethal myocarditis in transgenic mice

BACKGROUND Tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine with negative inotropic effects, can be detected in myocardium with end-stage heart failure, after endotoxin administration, and during transplant rejection. Various studies suggest that TNF-alpha participates in the pathogenesis of cardiac dysfunction. To test this hypothesis, transgenic mice were made that selectively overexpress TNF-alpha in cardiomyocytes. METHODS AND RESULTS A transgene construct was made containing the murine alpha-myosin heavy chain promoter and the coding sequence of murine TNF-alpha, followed by the simian virus 40 T-antigen intron and polyadenylation signals. Injection of this construct into fertilized eggs yielded three transgenic mice, all of which died spontaneously before the completion of weaning. Gross pathologic analysis of these mice demonstrated a decrease in body weight with markedly increased heart weight. Histologic examination of the heart revealed a substantial, diffuse lymphohistiocytic inflammatory infiltrate, associated with interstitial edema. Reverse transcriptase polymerase chain reaction showed that the transgene was expressed in the heart. Enzyme-linked immunosorbent assay demonstrated a substantial amount of TNF-alpha protein in the transgenic heart. CONCLUSION Overexpression of TNF-alpha in the heart leads to severe myocarditis and cardiomegaly. These results support the hypothesis that myocardial expression of TNF-alpha can contribute to the pathogenesis of cardiac dysfunction.

Proinflammatory cytokines regulate tissue inhibitors of metalloproteinases and disintegrin metalloproteinase in cardiac cells

OBJECTIVE Tissue inhibitors of metalloproteinases (TIMPs) are downregulated in the failing human heart. The objective of the present study was to test the hypothesis that cytokines might be involved in the regulation of TIMPs in cardiac cells. METHODS Neonatal Sprague-Dawley rat ventricular cells were exposed to 100 units/ml tumor necrosis factor-alpha and/or 5 ng/ml interleukin-1 beta. The mRNA and protein expression of TIMPs-1-4 and disintegrin metalloproteinase was analyzed using Northern blot, ELISA and/or Western blot, respectively. Proteolytic activity and extracellular matrix degradation and turnover were determined using gelatin zymography and pulse-chase experiments. RESULTS The TIMP-1 mRNA was upregulated in cardiac cells, while TIMP-1 protein levels were unchanged in myocytes but downregulated in non-myocytes. The TIMP-2 expression did not change with the cytokine treatment. TIMP-3 was downregulated at both the mRNA and protein levels in cardiac cells. TIMP-4 protein was transiently increased and then returned to control level. In contrast, disintegrin metalloproteinase mRNA and protein were significantly upregulated in those cells. The gelatinolytic activity and extracellular matrix protein degradation were significantly increased. CONCLUSIONS Tumor necrosis factor-alpha and interleukin-1 beta regulate the expression of TIMPs and disintegrin metalloproteinase, which may in turn contribute to the increased matrix degradation in cardiac cells. Since heart failure in humans is characterized by both re-expression of myocardial cytokines and remodeling of the extracellular matrix, those in vitro results suggest a potential role for those cytokines in the regulation of extracellular matrix remodeling and therefore in the transition to the end-stage heart failure phenotype.

Soluble Tumor Necrosis Factor Receptor Abrogates Myocardial Inflammation but Not Hypertrophy in Cytokine-Induced Cardiomyopathy

BACKGROUND Transgenic mice with cardiac-specific overexpression of tumor necrosis factor (TNF)-alpha develop dilated cardiomyopathy. The present study was designed to evaluate therapeutic effects of adenovirus-mediated neutralization of TNF-alpha on this model. METHODS AND RESULTS An adenovirus encoding the 55-kDa TNF receptor-IgG fusion protein (AdTNFRI) was injected intravenously into 6-week-old transgenic mice, which resulted in high levels of TNFRI in both plasma and myocardium. AdTNFRI did not reverse cardiomegaly but abrogated myocardial inflammation. Furthermore, AdTNFRI blocked the myocardial expression of intercellular adhesion molecule-1 and downstream cytokines, including interleukin-1beta and monocyte chemotactic protein-1. Downregulation of alpha-myosin heavy chain was restored by the treatment, whereas upregulation of beta-myosin heavy chain was not reversed. In contrast, the downregulation of sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban was normalized by AdTNFRI. Echocardiographic measurements showed that left ventricular end-systolic diameter was significantly larger in transgenic mice than in control mice, and this increase was reversed by the AdTNFRI treatment. However, left ventricular wall thickening was not reversed. CONCLUSIONS These results suggest that anti-TNF therapy may hold promise in the treatment of end-stage heart failure.

Sex-related survival differences in murine cardiomyopathy are associated with differences in TNF-receptor expression.

Epidemiological evidence suggests that the prognosis of heart failure in women is better than in men. In our murine model of dilated cardiomyopathy arising from cardiac-specific overexpression of TNF-alpha, the 6-month survival rate was significantly better in females than in males. Young female transgenic mice exhibited left ventricular wall thickening without dilatation, whereas age-matched male transgenic hearts were markedly dilated. Basal and isoproterenol-stimulated fractional shortening was preserved in female transgenic mice, but not in male transgenic mice. Myocardial expression of proinflammatory cytokines and the extent of myocardial infiltrates were similar in male and female transgenic mice. Myocardial expression of TNF-receptor mRNAs (type I and type II) was significantly higher in male mice in both transgenic and wild-type littermates, whereas sex-specific differences were not observed in either peripheral white blood cells or liver tissue. After TNF-alpha challenge, myocardial but not liver production of ceramide was significantly higher in male than in female mice. Thus, differential expression of myocardial TNF receptors may contribute to sex differences in the severity of congestive heart failure and mortality consequent to cardiac-specific overexpression of TNF-alpha.

Tumor Necrosis Factor Receptors 1 and 2 Differentially Regulate Survival, Cardiac Dysfunction, and Remodeling in Transgenic Mice With Tumor Necrosis Factor-α–Induced Cardiomyopathy

Background—Tumor necrosis factor (TNF)-&agr; plays a pathophysiological role in heart failure. Although both TNF receptor 1 (TNFR1) and 2 (TNFR2) are present in the heart, comparatively little is known about the role of TNFR2. Methods and Results—We bred TNFR1-knockout (KO) or TNFR2KO mice to transgenic (TG) mice with cardiac-specific overexpression of TNF-&agr; and analyzed resultant progeny. Six groups of male and female mice were studied: wild type (WT) with wild receptors (WT/W), TG with wild receptors (TG/W), TG with heterozygous receptor KO (TG/R1+/− or TG/R2+/−), and TG with homozygous receptor KO (TG/R1−/− or TG/R2−/−). Both male and female TG mice displayed cardiac hypertrophy, dilation, and reduced cardiac function. Male TG mice were more severely affected than genotypically matched females and died of heart failure at a younger age. Survival, cardiac function, and remodeling of TG/R1+/− and TG/R1−/− mice were improved relative to TG/W mice in both males and females. However, the survival of female TG/R2+/− and TG/R2−/− mice was worse than that of TG/W mice, with increased left ventricular dimension and left ventricular weight/body weight ratios. The cardiac TNF-&agr; protein level was upregulated in TG/R1−/− and TG/R2−/− compared with TG/W mice, whereas the level of TNF receptors was not downregulated in TG/W relative to WT/W mice. Conclusions—Ablation of the TNFR2 gene exacerbates heart failure and reduces survival, whereas ablation of TNFR1 blunts heart failure and improves survival. Signaling via TNFR2 may play a cardioprotective role in the pathogenesis of cytokine-mediated heart failure.

Adenosine Inhibits Lipopolysaccharide-Induced Cardiac Expression of Tumor Necrosis Factor-α

Tumor necrosis factor-alpha (TNF-alpha) is elevated in the failing heart. Very little is known about regulation of TNF-alpha in cardiomyocytes. TNF-alpha expression by macrophages is diminished by adenosine. Therefore, we hypothesized that a similar mechanism might occur in the heart. Neonatal rat myocytes were stimulated with lipopolysaccharide (LPS), and TNF-alpha was measured by ELISA. In the absence of LPS, myocytes did not release TNF-alpha in the medium. After exposure to LPS, TNF-alpha increased to 70.1+/-3.5 pg/mL at 6 hours. Immunofluorescent staining confirmed that TNF-alpha was expressed in myocytes. Adenosine decreased TNF-alpha in a dose-dependent manner (1 to 100 micromol/L, 37% to 65% decrease, P<.01). Adenosine also decreased TNF-alpha in cell homogenates by 78% (P<.0001). The effect of adenosine could be replicated by the A2 agonist PD-125944 (DPMA), by cAMP agonists 8-bromo-cAMP, forskolin, and Ro 20-1724, but not by A1 and A3 agonists. Conversely, the effect of adenosine could be suppressed by the adenylate cyclase inhibitor MDL-12,330. Adenosine also inhibited TNF-alpha in adult rat ventricular myocytes (-60%, P<.005) and rat papillary muscles (-55%, P<.05). In neonatal myocytes, adenosine normalized LPS-induced calcium changes and improved LPS-induced negative inotropic (P<.01) and negative lusitropic (P<.01) effects. Our results demonstrate that adenosine can significantly diminish TNF-alpha levels in the heart. The effect appears to be mediated by the A2 receptor and transduced through a G protein-adenylyl cyclase pathway. These results may explain some cardioprotective effects of adenosine and provide a novel pharmacological intervention in congestive heart failure.

Expression of proinflammatory cytokines in the failing human heart: comparison of recent-onset and end-stage congestive heart failure

BACKGROUND Plasma levels of proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, are elevated in patients with congestive heart failure (CHF). Recent studies suggest that the failing human heart is a source of proinflammatory cytokines in the end-stage failing heart. However, the relevance of plasma levels to those of the myocardium remains undefined. We sought to compare cytokine expression in early and end-stage CHF, and to evaluate the correlation of tissue expression to plasma levels. METHODS Two patient populations were studied: patients with recent-onset CHF, all with symptoms less than 6 months (n = 17, duration of symptoms 2.1 +/- 1.6 months, range of New York Heart Association (NYHA) 1 to 3), and end-stage heart-failure patients (n = 7) who underwent left-ventricular assist-device (LVAD) implantation (Duration of symptoms 47.1 +/- 28.0 months, all NYHA class 4). Plasma levels of TNF-alpha and IL-6 proteins were evaluated by an Enzyme-Linked Immuno-Sorbent Assay (ELISA), while myocardial levels of cytokine transcripts were assessed by ribonuclease (Rnase) protection assay. RESULTS In patients with end-stage heart failure, TNF-alpha and IL-6 were increased in the plasma as well as in the myocardium (plasma: TNF-alpha = 7.7 +/- 2.3 pg/ml, IL-6 = 45.0 +/- 47.1 pg/ml; myocardium: TNF-alpha = 0.31 +/- 0.15% of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, IL-6 = 1.56 +/- 1.54% ). In contrast, despite elevated plasma levels of TNF-alpha and IL-6, the myocardium of patients with the recent onset of symptoms demonstrated minimal expression of TNF-alpha and IL-6 messenger ribonucleic acid (mRNA) (plasma: TNF-alpha = 4.3 +/- 1.7 pg/ml, IL-6 = 3.3 +/- 1.8 pg/ml; myocardium: TNF-alpha = 0.13 +/- 0. 04%, IL-6 = 0.02 +/- 0.04%). Plasma levels of TNF-alpha were significantly correlated with those in the myocardium when both populations were combined. (r = 0.69, p < 0.001). CONCLUSIONS Cytokines are expressed in the myocardium in end-stage heart failure to a much greater degree than in patients with the recent-onset of symptoms. This suggests that induction of cytokines in the myocardium is a relatively late event in the pathogenesis of CHF. Furthermore, plasma levels of TNF-alpha correlates with mRNA expression in the myocardium and thus may serve as an appropriate marker of myocardial cytokine activation. Whether the production of cytokines in the failing human heart precedes the elevation of cytokines in the plasma remains undefined. Therefore, we studied expression of TNF-alpha and IL-6 in the myocardium as well as in the plasma in patients with early and end-stage CHF. The results have demonstrated that cytokines are expressed in the myocardium in end-stage heart failure to a much greater degree than in patients with the recent onset of symptoms. This suggests that induction of cytokines in the myocardium is a relatively late event in the pathogenesis of CHF.