Dysferlin mediates the cytoprotective effects of TRAF2 following myocardial ischemia reperfusion injury.

BACKGROUND: We have demonstrated that tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), a scaffolding protein common to TNF receptors 1 and 2, confers cytoprotection in the heart. However, the mechanisms for the cytoprotective effects of TRAF2 are not known. METHODS/RESULTS: Mice with cardiac-restricted overexpression of low levels of TRAF2 (MHC-TRAF2LC) and a dominant negative TRAF2 (MHC-TRAF2DN) were subjected to ischemia (30-minute) reperfusion (60-minute) injury (I/R), using a Langendorff apparatus. MHC-TRAF2LC mice were protected against I/R injury as shown by a significant ≈27% greater left ventricular (LV) developed pressure after I/R, whereas mice with impaired TRAF2 signaling had a significantly ≈38% lower LV developed pressure, a ≈41% greater creatine kinase (CK) release, and ≈52% greater Evans blue dye uptake after I/R, compared to LM. Transcriptional profiling of MHC-TRAF2LC and MHC-TRAF2DN mice identified a calcium-triggered exocytotic membrane repair protein, dysferlin, as a potential cytoprotective gene responsible for the cytoprotective effects of TRAF2. Mice lacking dysferlin had a significant ≈39% lower LV developed pressure, a ≈20% greater CK release, and ≈29% greater Evans blue dye uptake after I/R, compared to wild-type mice, thus phenocopying the response to tissue injury in the MHC-TRAF2DN mice. Moreover, breeding MHC-TRAF2LC onto a dysferlin-null background significantly attenuated the cytoprotective effects of TRAF2 after I/R injury. CONCLUSION: The study shows that dysferlin, a calcium-triggered exocytotic membrane repair protein, is required for the cytoprotective effects of TRAF2-mediated signaling after I/R injury.

SRC-2 is an essential coactivator for orchestrating metabolism and circadian rhythm.

Synchrony of the mammalian circadian clock is achieved by complex transcriptional and translational feedback loops centered on the BMAL1:CLOCK heterodimer. Modulation of circadian feedback loops is essential for maintaining rhythmicity, yet the role of transcriptional coactivators in driving BMAL1:CLOCK transcriptional networks is largely unexplored. Here, we show diurnal hepatic steroid receptor coactivator 2 (SRC-2) recruitment to the genome that extensively overlaps with the BMAL1 cistrome during the light phase, targeting genes that enrich for circadian and metabolic processes. Notably, SRC-2 ablation impairs wheel-running behavior, alters circadian gene expression in several peripheral tissues, alters the rhythmicity of the hepatic metabolome, and deregulates the synchronization of cell-autonomous metabolites. We identify SRC-2 as a potent coregulator of BMAL1:CLOCK and find that SRC-2 targets itself with BMAL1:CLOCK in a feedforward loop. Collectively, our data suggest that SRC-2 is a transcriptional coactivator of the BMAL1:CLOCK oscillators and establish SRC-2 as a critical positive regulator of the mammalian circadian clock.

Tumor necrosis factor receptor-associated factor 2 signaling provokes adverse cardiac remodeling in the adult mammalian heart.

BACKGROUND: Tumor necrosis factor superfamily ligands provoke a dilated cardiac phenotype signal through a common scaffolding protein termed tumor necrosis factor receptor-associated factor 2 (TRAF2); however, virtually nothing is known about TRAF2 signaling in the adult mammalian heart. METHODS AND RESULTS: We generated multiple founder lines of mice with cardiac-restricted overexpression of TRAF2 and characterized the phenotype of mice with higher expression levels of TRAF2 (myosin heavy chain [MHC]-TRAF2(HC)). MHC-TRAF2(HC) transgenic mice developed a time-dependent increase in cardiac hypertrophy, left ventricular dilation, and adverse left ventricular remodeling, and a significant decrease in LV+dP/dt and LV-dP/dt when compared with littermate controls (P<0.05 compared with littermate). During the early phases of left ventricular remodeling, there was a significant increase in total matrix metalloproteinase activity that corresponded with a decrease in total myocardial fibrillar collagen content. As the MHC-TRAF2(HC) mice aged, there was a significant decrease in total matrix metalloproteinase activity accompanied by an increase in total fibrillar collagen content and an increase in myocardial tissue inhibitor of metalloproteinase-1 levels. There was a significant increase in nuclear factor-κB activation at 4 to 12 weeks and jun N-terminal kinases activation at 4 weeks in the MHC-TRAF2(HC) mice. Transciptional profiling revealed that >95% of the hypertrophic/dilated cardiomyopathy-related genes that were significantly upregulated genes in the MHC-TRAF2(HC) hearts contained κB elements in their promoters. CONCLUSIONS: These results show for the first time that targeted overexpression of TRAF2 is sufficient to mediate adverse cardiac remodeling in the heart.

The development of myocardial fibrosis in transgenic mice with targeted overexpression of tumor necrosis factor requires mast cell-fibroblast interactions.

BACKGROUND: Transgenic mice with cardiac-restricted overexpression of tumor necrosis factor (MHCsTNF mice) develop progressive myocardial fibrosis, diastolic dysfunction, and adverse cardiac remodeling. Insofar as tumor necrosis factor (TNF) does not directly stimulate fibroblast collagen synthesis, we asked whether TNF-induced fibrosis was mediated indirectly through interactions between mast cells and cardiac fibroblasts. METHODS AND RESULTS: Cardiac mast cell number increased 2 to 3 fold (P<0.001) in MHCsTNF mice compared with littermate controls. Outcrossing MHCsTNF mice with mast cell-deficient (c-kit(-/-)) mice showed that the 11-fold increase (P<0.001) in collagen volume fraction in MHCsTNF/c-kit(+/-) mice was abrogated in MHCsTNF/c-kit(-/-) mice, and that the leftward shifted left ventricular pressure-volume curve in the MHCsTNF/c-kit(+/-) mice was normalized in the MHCsTNF/c-kit(-/-) hearts. Furthermore, the increase in transforming growth factor ß1 and type I transforming growth factor ß receptor messenger RNA levels was significantly (P=0.03, P=0.01, respectively) attenuated in MHCsTNF/c-kit(-/-) when compared with MHCsTNF/c-kit(+/-) mice. Coculture of fibroblasts with mast cells resulted in enhanced α-smooth muscle actin expression, increased proliferation and collagen messenger RNA expression, and increased contraction of 3-dimensional collagen gels in MHCsTNF fibroblasts compared with littermate fibroblasts. The effects of mast cells were abrogated by type I transforming growth factor ß receptor antagonist NP-40208. CONCLUSIONS: These results suggest that increased mast cell density with resultant mast cell-cardiac fibroblast cross-talk is required for the development of myocardial fibrosis in inflammatory cardiomyopathy. Cardiac fibroblasts exposed to sustained inflammatory signaling exhibit an increased repertoire of profibrotic phenotypic responses in response to mast cell mediators.

The cytoprotective effects of tumor necrosis factor are conveyed through tumor necrosis factor receptor-associated factor 2 in the heart.

BACKGROUND: Activation of both type 1 and type 2 tumor necrosis factor (TNF) receptors (TNFR1 and TNFR2) confers cytoprotection in cardiac myocytes. Noting that the scaffolding protein TNF receptor-associated factor 2 (TRAF2) is common to both TNF receptors, we hypothesized that the cytoprotective responses of TNF were mediated through TRAF2. METHODS AND RESULTS: Mice with cardiac-restricted overexpression of low levels of TNF (MHCsTNF(3)) and TRAF2 (MHC-TRAF2(LC)) and mice lacking TNFR1, TNFR2, and TNFR1/TNFR2 were subjected to ischemia (30 minutes) reperfusion (30 minutes) injury ex vivo using a Langendorff apparatus. MHCsTNF(3) mice were protected against ischemia-reperfusion injury as shown by a significant approximately 30% greater left ventricular developed pressure, approximately 80% lower creatine kinase release, and Evans blue dye uptake compared with littermates. The extent of ischemia-reperfusion induced injury was similar in wild-type, TNFR1, and TNFR2 deficient mice; however, mice lacking TNFR1/TNFR2 had a significant approximately 40% lower left ventricular developed pressure, a approximately 65% greater creatine kinase release, and approximately 40% greater Evans blue dye uptake compared with littermates. Interestingly, MHC-TRAF2(LC) mice had a significant approximately 50% lower left ventricular developed pressure, a approximately 70% lower creatine kinase release, and approximately 80% lower Evans blue dye uptake compared with littermate controls after ischemia-reperfusion injury. Biochemical analysis of the MHC-TRAF2(LC) hearts showed that there was activation of nuclear factor-kappaB but not c-Jun N-terminal kinase activation. CONCLUSIONS: Taken together, these results suggest that TNF confers cytoprotection in the heart through TRAF2-mediated activation of nuclear factor-kappaB.

Adaptive and maladptive effects of SMAD3 signaling in the adult heart after hemodynamic pressure overloading.

BACKGROUND: Previous studies suggest that transforming growth factor-beta provokes cardiac hypertrophy and myocardial fibrosis; however, it is unclear whether the deleterious effects of transforming growth factor-beta signaling are conveyed through SMAD-dependent or SMAD-independent signaling pathways. METHODS AND RESULTS: To determine the contribution of SMAD-dependent signaling to cardiac remodeling, we performed transaortic constriction in SMAD3 null (SMAD3(-/-)) and littermate control mice (age, 10 to 12 weeks). Cumulative survival 20 days after transaortic constriction was significantly less in the SMAD3(-/-) mice when compared with littermate controls (43.6% versus 90.9%, P<0.01). Transaortic constriction resulted in a significant increase in cardiac hypertrophy in the SMAD3(-/-) mice, denoted by an increase in the heart weight to tibial length ratio and increased myocyte cross-sectional area. Loss of SMAD3 signaling also resulted in a significant 60% decrease in myocardial fibrosis (P<0.05). A microRNA microarray showed that 55 microRNAs were differentially expressed in littermate and SMAD3(-/-) mice and that 10 of these microRNAs were predicted to bind to genes that regulate the extracellular matrix. Of these 10 candidate microRNAs, both miR-25 and miR-29a were sufficient to decrease collagen gene expression when transfected into isolated cardiac fibroblasts in vitro. CONCLUSIONS: The results suggest that SMAD3 signaling plays dual roles in the heart: one beneficial role by delimiting hypertrophic growth and the other deleterious by modulating myocardial fibrosis, possibly through a pathway that entails accumulation of microRNAs that decrease collagen gene expression.

Oxidative stress promotes ligand-independent and enhanced ligand-dependent tumor necrosis factor receptor signaling.

Tumor necrosis factor (TNF) receptor 1 (TNFR1, p55) and 2 (TNFR2, p75) are characterized by several cysteine-rich modules in the extracellular domain, raising the possibility that redox-induced modifications of these cysteine residues might alter TNFR function. To test this possibility, we examined fluorescence resonance energy transfer (FRET) in 293T cells transfected with CFP- and YFP-tagged TNFRs exposed to the thiol oxidant diamide. Treatment with high concentrations of diamide (1 mm) resulted in an increase in the FRET signal that was sensitive to inhibition with the reducing agent dithiothreitol, suggesting that oxidative stress resulted in TNFR self-association. Treatment of cells with low concentrations of diamide (1 mum) that was not sufficient to provoke TNFR self-association resulted in increased TNF-induced FRET signals relative to the untreated cells, suggesting that oxidative stress enhanced ligand-dependent TNFR signaling. Similar findings were obtained when the TNFR1- and TNFR2-transfected cells were pretreated with a cell-impermeable oxidase, DsbA, that catalyzes disulfide bond formation between thiol groups on cysteine residues. The changes in TNFR self-association were functionally significant, because pretreating the HeLa cells and 293T cells resulted in increased TNF-induced NF-kappaB activation and TNF-induced expression of IkappaB and syndecan-4 mRNA levels. Although pretreatment with DsbA did not result in an increase in TNF binding to TNFRs, it resulted in increased TNF-induced activation of NF-kappaB, consistent with an allosteric modification of the TNFRs. Taken together, these results suggest that oxidative stress promotes TNFR receptor self-interaction and ligand-independent and enhanced ligand-dependent TNF signaling.

Targeted gene silencing of tumor necrosis factor attenuates the negative inotropic effects of lipopolysaccharide in isolated contracting cardiac myocytes.

Bacterial endotoxin (lipopolysaccharide) depresses cardiovascular function; however, the mediators and signaling pathways that are responsible for the negative inotropic effects of lipopolysaccharide are not fully known. We used RNA interference to determine the relative role of tumor necrosis factor with respect to mediating the negative inotropic effects of lipopolysaccharide in isolated cardiac myocytes. Cardiac myocyte cultures were treated with lipopolysaccharide in the presence or absence of small interfering RNAs (siRNA) for tumor necrosis factor. We examined the effects of tumor necrosis factor siRNA on lipopolysaccharide-induced tumor necrosis factor messenger RNA (mRNA) and protein biosynthesis, as well as the negative inotropic effects of lipopolysaccharide in isolated contracting cardiac myocytes. Treatment of adult cardiac myocyte cultures with tumor necrosis factor siRNA significantly attenuated lipopolysaccharide-induced tumor necrosis factor mRNA and protein biosynthesis, whereas transfection with a double-stranded RNA that does not target mammalian mRNA had no effect. Pretreatment with tumor necrosis factor siRNA significantly attenuated, but did not abrogate, the lipopolysaccharide-induced decrease in sarcomere shortening in isolated contracting cardiac myocytes. In contrast, tumor necrosis factor siRNA had a comparatively smaller effect on improving sarcomere shortening once the negative inotropic effects of lipopolysaccharide were fully established. These results suggest that tumor necrosis factor plays an important upstream role in lipopolysaccharide-induced negative inotropic effects in isolated contracting cardiac myocytes and that other molecular mechanisms are responsible for the decrease in sarcomere shortening after sustained lipopolysaccharide signaling.

Effect of the soluble TNF-antagonist etanercept on tumor necrosis factor bioactivity and stability.

BACKGROUND: Targeted anti-tumor necrosis factor (TNF) strategies in patients with rheumatoid arthritis have resulted in new and/or worsening heart failure in individuals who were free of cardiovascular disease. METHODS AND RESULTS: To determine the mechanism of new and/or worsening heart failure in patients who were receiving the soluble TNF-antagonist etanercept, we analyzed frozen plasma samples from a previous clinical trial with etanercept in heart failure patients, and conducted complimentary mechanistic in vitro studies. Analysis of the clinical trial data showed that use of etanercept resulted in a significant 70-fold increase in the level of immunoreactive TNF. Complimentary in vitro studies using an L929 bioassay showed that at low concentrations of etanercept relative to TNF there was an unexpected 1.5- to 1.75-fold increase in the absolute level of TNF bioactivity. We also examined the effect of etanercept on TNF stability and the results showed that there was a two-fold increase in the mass of bioactive homotrimeric TNF when the molar ratio of TNF to etanercept was approximately 200:1. CONCLUSION: Etanercept increases the immunoreactive mass of TNF in heart failure patients, as well as augments TNF cytotoxicity in certain settings, thus suggesting one potential mechanism for the worsening heart failure in some patients who were receiving this agent.

Transforming growth factor-beta receptor antagonism attenuates myocardial fibrosis in mice with cardiac-restricted overexpression of tumor necrosis factor.

The mechanisms that are responsible for the development of myocardial fibrosis in inflammatory cardiomyopathy are unknown. We have previously generated lines of transgenic mice with cardiac-restricted overexpression of tumor necrosis factor (MHCsTNF mice), a pro-inflammatory cytokine. The MHCsTNF mice develop a heart failure phenotype that is characterized by progressive myocardial fibrosis, as well as increased levels transforming growth factor-beta (TGF-beta)(mRNA and protein. In order to determine whether TGF-beta-mediated signaling was responsible for the myocardial fibrosis observed in the MHCsTNF mice, we treated MHCsTNF and littermate control mice from 4 to 12 weeks of age with a novel orally available TGF-beta receptor antagonist (NP-40208). At the time of terminal study, myocardial collagen content was determined using the picrosirius red technique, and left ventricular (LV) systolic and diastolic function were determined using the Langendorff method. Treatment with NP-40208 resulted in a significant (P < 0.05) 65% decrease in nuclear translocation of Smad 2/3, a significant (P < 0.05), decrease in the heart-weight to body-weight ratio from 6.5 to 5.7, a approximately 37% decrease in fibrillar collagen content (P < 0.01) and a significant (P < 0.05) decrease in the LV chamber stiffness by approximately 25% in the MHCsTNF mice when compared to diluent-treated controls. Treatment with NP-40208 had no discernable effect on LV systolic function, nor any effect on cardiac myocyte size or fetal gene expression in the MHCsTNF mice. Taken together, these observations suggest that sustained pro-inflammatory signaling in the adult heart is associated with a pro-fibrotic phenotype that arises, at least in part, from TGF-beta-mediated signaling, with resultant activation of Smad 2/3, leading to increased myocardial fibrosis and increased LV diastolic chamber stiffness.

Downregulation of connexin40 and increased prevalence of atrial arrhythmias in transgenic mice with cardiac-restricted overexpression of tumor necrosis factor.

Atrial arrhythmias, primarily atrial fibrillation, have been independently associated with structural remodeling and with inflammation. We hypothesized that sustained inflammatory signaling by tumor necrosis factor (TNF) would lead to alterations both in underlying atrial myocardial structure and in atrial electrical conduction. We performed ECG recording, intracardiac electrophysiology studies, epicardial mapping, and connexin immunohistochemical analyses on transgenic mice with targeted overexpression of TNF in the cardiac compartment (MHCsTNF) and on wild-type (WT) control mice (age 8-16 wk). Atrial and ventricular conduction abnormalities were always evident on ECG in MHCsTNF mice, including a shortened atrioventricular interval with a wide QRS duration secondary to junctional rhythm. Supraventricular arrhythmias were observed in five of eight MHCsTNF mice, whereas none of the mice demonstrated ventricular arrhythmias. No arrhythmias were observed in WT mice. Left ventricular conduction velocity during apical pacing was similar between the two mouse groups. Connexin40 was significantly downregulated in MHCsTNF mice. In contrast, connexin43 density was not significantly altered in MHCsTNF mice, but rather dispersed away from the intercalated disks. In conclusion, sustained inflammatory signaling contributed to atrial structural remodeling and downregulation of connexin40 that was associated with an increased prevalence of atrial arrhythmias.

Myotrophin/V-1 does not act as an extracellular signal to induce myocyte hypertrophy.

The myotrophin/V-1 protein was originally found to be elevated in failing heart tissues and was described as an exogenously acting hypertrophy-inducing factor. However, several studies have proposed only intracellular functions for this protein. We investigated whether this protein is an exogenously acting hypertrophy-inducing trophin or an intracellular nuclear factor of kappa B (NFkappaB) regulatory protein. In the current report, immunofluorescence and cell fractionation studies showed that myotrophin is present only in the cytoplasm and is not actively released into the extracellular environment in response to hypertrophy-inducing stimuli. Moreover, in response to ischemia/reperfusion injury, an active release of myotrophin from adult rat myocardium was not observed. Furthermore, protein synthesis studies in rat neonatal myocytes indicated that exogenous myotrophin did not induce hypertrophy. On the other hand, myotrophin stimulates the generation of NFkappaB dimers in vitro and thus regulates the NFkappaB-mediated transcription in cardiac myocytes. Taken together, these studies suggest that myotrophin is a strictly cytosolic protein that regulates the NFkappaB-mediated transcriptional process.

Heterogeneous effects of tissue inhibitors of matrix metalloproteinases on cardiac fibroblasts.

The balance between matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), plays a critical role in cardiac remodeling. Although a number of studies have characterized the pathophysiological role of MMPs in the heart, very little is known with respect to the role of TIMPs in the heart. To delineate the role of TIMPs in the heart we examined the effects of adenovirus-mediated overexpression of TIMP-1, -2, -3, and -4 in cardiac fibroblasts. Infection of cardiac fibroblasts with adenoviral constructs containing human recombinant TIMP (AdTIMP-1, -2, -3, and -4) provoked a significant (P < 0.0001) 1.3-fold in increase in bromodeoxyuridine (BrdU) incorporation. Similarly, treatment of cardiac fibroblasts with AdTIMP-1-, -2-, -3-, and -4-conditioned medium led to a 1.2-fold increase in BrdU incorporation (P < 0.0001) that was abolished by pretreatment with anti-TIMP-1, -2, -3, and -4 antibodies. The effects of TIMPs were not mimicked by treating the cells with RS-130830, a broad-based MMP inhibitor, suggesting that the effects of TIMPs were independent of their ability to inhibit MMPs. Infection with AdTIMP-1, -2, -3, and -4 led to a significant increase in alpha-smooth muscle actin staining, consistent with TIMP-induced phenotypic differentiation into myofibroblasts. Finally, infection with AdTIMP-2 resulted in a significant increase in collagen synthesis, whereas infection with AdTIMP-3 resulted in a significant increase in fibroblast apoptosis. TIMPs exert overlapping as well as diverse effects on isolated cardiac fibroblasts. The observation that TIMPs stimulate fibroblast proliferation as well as phenotypic differentiation into myofibroblasts suggests that TIMPs may play an important role in tissue repair in the heart that extends beyond their traditional role as MMP inhibitors.

Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression.

Although cardiac myocyte apoptosis has been detected in explanted hearts from patients with end-stage dilated and ischemic cardiomyopathy, the relative contribution of apoptotic cell death to left ventricular (LV) remodeling and cardiac decompensation is not known. To determine whether progressive cardiac myocyte apoptosis contributes to the transition from a hypertrophic to a dilated cardiac phenotype that is observed in transgenic myosin heavy chain secreted TNF (MHCsTNF) mice with cardiac restricted overexpression of tumor necrosis factor (TNF), we assessed cardiac myocyte apoptosis (using a DNA ligase technique) in MHCsTNF mice and littermate control mice in relation to serial changes in LV structure, which was assessed using MRI. The prevalence of cardiac myocyte apoptosis increased progressively from 4 to 12 wk as the hearts of the MHCsTNF mice underwent the transition from a concentric hypertrophic to a dilated cardiac phenotype. Treatment of the MHCsTNF mice with the broad-based caspase inhibitor N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino4-oxo-5-fluoropentanoic acid significantly decreased cardiac myocyte apoptosis and significantly attenuated LV wall thinning and adverse cardiac remodeling. Additional studies suggested that the TNF-induced decrease in Bcl-2 expression and activation of the intrinsic mitochondrial death pathway were responsible for the cardiac myocyte apoptosis observed in the MHCsTNF mice. These studies show that progressive cardiac myocyte apoptosis is sufficient to contribute to adverse cardiac remodeling in the adult mammalian heart through progressive LV wall thinning.

Cross-regulation between the renin-angiotensin system and inflammatory mediators in cardiac hypertrophy and failure.

One of the major conceptual advances in our understanding of the pathogenesis of heart failure has been the insight that heart failure may progress as the result of the sustained overexpression of biologically active "neurohormones", such as norepinephrine and angiotensin II, which by virtue of their deleterious effects are sufficient to contribute to disease progression by provoking worsening left ventricular (LV) remodeling and progressive LV dysfunction. Recently, a second class of biologically active molecules, termed cytokines, has also been identified in the setting of heart failure. Analogous to the situation with neurohormones, the overexpression of cytokines is sufficient to contribute to disease progression in heart failure phenotype. Although important interactions between proinflammatory cytokines and the adrenergic system have been recognized in the heart for over a decade, the nature of the important interactions between proinflammatory cytokines and the renin-angiotensin system has become apparent only recently. Accordingly, in the present review, we will discuss the evidence which suggests that there is a functionally significant cross-talk between neurohormonal and inflammatory cytokine signaling in cardiac hypertrophy and failure.

Cyclooxygenase-2 inhibitor treatment improves left ventricular function and mortality in a murine model of doxorubicin-induced heart failure.

BACKGROUND: Progression of heart failure after initial myocardial injury is mediated in part by various redundant inflammatory mediators, including the widely expressed cyclooxygenase-2 (COX-2). Because COX-2 inhibitors are useful in treating many inflammation-mediated diseases, we asked whether COX-2 inhibition can attenuate heart failure progression. METHODS AND RESULTS: Heart failure was experimentally induced in 100 mice by administration of doxorubicin (4 mg. kg(-1). wk(-1) for 6 weeks). Beginning at day 42, mice were fed daily with either COX-2 inhibitor-containing mice chow (n=50) or plain mice chow (controls; n=50). Left ventricular ejection fraction was evaluated as a measure of heart failure by a novel method of transthoracic echocardiography (with intravascular ultrasound catheters) at baseline and on days 42, 56, and 70. From baseline to study termination, left ventricular ejection fraction in COX-2 inhibitor-treated mice decreased significantly less than in control mice (9% versus 29%, P<0.01). Mortality was significantly lower for COX-2 inhibitor-treated mice than for control mice (18% versus 38%, P<0.01). These results were confirmed in a revalidation study in COX-2 inhibitor-treated mice (n=25) and controls (n=25). That study revealed that the hearts from control mice weighed roughly the same as hearts from COX-2 inhibitor-treated mice but showed more extensive signs of cardiomyopathy (as determined by pathological analysis by an independent, blinded observer) and higher levels of COX-2 proteins (as determined by immunoblotting [6442+/-1635 versus 4300+/-2408 arbitrary units, P<0.022]). CONCLUSIONS: COX-2 inhibitors can attenuate the progression of heart failure in a murine model of doxorubicin-induced heart failure.

Targeted overexpression of noncleavable and secreted forms of tumor necrosis factor provokes disparate cardiac phenotypes.

BACKGROUND: Recent studies suggest that posttranslation processing or "shedding" (ie, secretion) of tumor necrosis factor (TNF) by tumor necrosis factor-alpha converting enzyme (TACE) may contribute to the left ventricular (LV) remodeling that occurs in the failing human heart. METHODS AND RESULTS: To address the functional significance of TNF shedding, we generated lines of transgenic mice with targeted overexpression of secreted wild-type (MHCsTNF2) TNF and overexpression of a mutated noncleavable transmembrane form of TNF (MHCmTNF). Both lines of mice had overlapping levels of myocardial TNF protein; however, the phenotypes of the MHCsTNF2 and MHCmTNF mice were strikingly disparate. Whereas the MHCmTNF mice developed a concentric LV hypertrophy phenotype, the MHCsTNF2 mice developed a dilated LV phenotype. The fibrillar collagen weave in MHCmTNF mice with concentric hypertrophy was characterized by thick collagen fibrils and increased collagen content, whereas the fibrillar collagen weave in the MHCsTNF2 mice with LV dilation was characterized by a diminished collagen content. Inhibition of matrix metalloproteinases with a broad-based matrix metalloproteinase inhibitor prevented LV dilation in the MHCsTNF2 mice. CONCLUSIONS: These findings suggest that posttranslational processing of TNF, as opposed to TNF expression per se, is responsible for the adverse cardiac remodeling that occurs after sustained TNF overexpression.

Nuclear factor-kappaB protects the adult cardiac myocyte against ischemia-induced apoptosis in a murine model of acute myocardial infarction.

BACKGROUND: Previous studies have shown that tumor necrosis factor (TNF) confers cytoprotective responses in cardiac myocytes. However, the mechanisms for the cytoprotective effects of TNF remain unknown. Given that TNF signals through nuclear factor kappaB (NF-kappaB) and given that NF-kappaB mediates cytoprotective responses, we asked whether NF-kappaB activation conferred cytoprotective responses in acute myocardial ischemia/infarction. METHODS AND RESULTS: We examined infarct size and the prevalence of apoptosis in transgenic mice harboring cardiac-restricted expression of a mutated IkappaBalpha protein (IkappaBalphaDeltaN) that prevents nuclear translocation of NF-kappaB in cardiac myocytes. Triphenyltetrazolium chloride staining showed that infarct size was approximately 50% greater (P<0.02) in the IkappaBalphaDeltaN mice compared with littermate controls at 24 hours. The prevalence of cardiac myocyte apoptosis was significantly greater (P<0.008) in the IkappaBalphaDeltaN mice compared with the littermate control mice 3 and 6 hours after left anterior descending occlusion. To explore the mechanism for these findings, we examined protein levels of c-IAP1, c-IAP2, and Bcl-2 as well as manganese superoxide dismutase and c-Jun NH2-terminal kinase activity. These studies showed that protein levels of c-IAP1 and Bcl-2 were significantly lower in the IkappaBalphaDeltaN mice, whereas there was no change in c-IAP2 levels, manganese superoxide dismutase, or c-Jun NH2-terminal kinase activity. CONCLUSIONS: Transgenic mice with a defect in activation of NF-kappaB have increased susceptibility to tissue injury after acute left anterior descending occlusion. These studies suggest that the cytoprotective effects of NF-kappaB are mediated, at least in part, by Bcl-2 or c-IAP1.

Targeted overexpression of transmembrane tumor necrosis factor provokes a concentric cardiac hypertrophic phenotype.

BACKGROUND: Tumor necrosis factor (TNF) is initially synthesized as a 26-kDa transmembrane protein that is enzymatically cleaved by TNF-alpha converting enzyme (TACE) to generate a 17-kDa form of "secreted" TNF. Whereas the effects of secreted TNF in the heart have been characterized extensively, the effects of transmembrane TNF in the heart are unknown. METHODS AND RESULTS: We generated lines of transgenic mice with cardiac-restricted overexpression of a noncleavable, transmembrane form of TNF. We next treated a previously generated transgenic line of mice with cardiac-restricted expression of cleavable TNF (referred to as MHCsTNF mice) with a TACE inhibitor (DPC-IDR1) to determine whether TACE inhibition would prevent the transition from concentric hypertrophy to left ventricular (LV) dilation that occurs in this line of transgenic mice. Two of the founder lines did not have a demonstrable phenotype (M-41 and M-45), whereas a third line developed a concentric hypertrophic cardiac phenotype (M-48). Characterization of the M-48 line at 6 weeks of age showed that this line developed concentric hypertrophy, with an increase in myocyte cross-sectional area and reexpression of the fetal gene program. Four weeks of TACE inhibition abrogated the LV dilation in the MHCsTNF mice and resulted in an increase in LV wall thickness and increased myocyte cross-sectional area, thus mimicking the effects observed in the mice with noncleavable, transmembrane TNF. CONCLUSIONS: These studies show that transmembrane TNF is biologically active and provokes a concentric hypertrophic cardiac phenotype, thus suggesting that posttranslational processing (ie, secretion) of TNF is responsible for the dilated cardiomyopathic phenotype in mice with targeted, cardiac-restricted overexpression of TNF.

Activation and functional significance of the renin-angiotensin system in mice with cardiac restricted overexpression of tumor necrosis factor.

BACKGROUND: The functional significance of cross-regulation between the renin-angiotensin system (RAS) and tumor necrosis factor (TNF) has been established in nonmyocyte cell types; however, the degree and functional significance of the interaction between RAS and TNF has not been characterized in the heart. METHODS AND RESULTS: We examined the expression of components of the RAS in a line of transgenic mice (MHCsTNF) with cardiac restricted overexpression of TNF. When examined at 4, 8, and 12 weeks of age, the MHCsTNF mice had increased activation of myocardial RAS, as shown by an increase in ACE mRNA level and ACE activity and increased angiotensin II peptide levels. Furthermore, myocardial angiotensin receptor mRNA and protein levels were reduced in the MHCsTNF mice, consistent with homologous desensitization of the receptors. However, expression of renin and angiotensinogen was not increased in MHCsTNF mice compared with littermate controls. To determine the functional significance of RAS activation in the MHCsTNF mice, we treated the mice with an angiotensin type I receptor antagonist, losartan (30 mg/kg), or diluent from 4 to 8 weeks of age. Analysis of cardiac structure with MRI showed that treatment with losartan normalized left ventricular mass and wall thickness. Furthermore, treatment with losartan reduced myocardial collagen content and reduced the incidence of myocyte apoptosis. CONCLUSIONS: Taken together, these results show that there are functionally significant interactions between RAS and TNF in the heart and that these interactions play an important role in the development and progression of left ventricular remodeling.