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.

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.

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.

Angiotensin II induces tumor necrosis factor biosynthesis in the adult mammalian heart through a protein kinase C-dependent pathway.

BACKGROUND: Previous studies suggest that angiotensin II (Ang II) upregulates the expression of tumor necrosis factor (TNF) in nonmyocyte cell types; however, the effect of Ang II on TNF expression in the adult mammalian heart is not known. METHODS AND RESULTS: To determine whether Ang II was sufficient to provoke TNF biosynthesis in the adult heart, we examined the effects of Ang II in isolated buffer-perfused Langendorff feline hearts. Ang II (10(-7) mol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF mRNA and protein biosynthesis in the heart as well as in cultured adult cardiac myocytes. The effects of Ang II on myocardial TNF mRNA and protein synthesis were mediated through the angiotensin type 1 receptor (AT1R), insofar as an AT1R antagonist (AT1a) blocked the effects of Ang II, whereas an angiotensin type 2 receptor (AT2R) antagonist (AT2a) had no effect. Stimulation with Ang II led to the activation of nuclear factor-kappaB and activator protein-1 (AP-1), two transcription factors that are important for TNF gene expression. Nuclear factor-kappaB activation was accompanied by phosphorylation of IkappaBalpha on serine 32 as well as degradation of IkappaBalpha, suggesting that the effects of Ang II were mediated through an IkappaBalpha-dependent pathway. The important role of protein kinase C (PKC) was suggested by studies in which a phorbol ester triggered TNF biosynthesis, and a PKC inhibitor abrogated Ang II-induced TNF biosynthesis. CONCLUSIONS: These studies suggest that Ang II provokes TNF biosynthesis in the adult mammalian heart through a PKC-dependent pathway.

Load-dependent and -independent regulation of proinflammatory cytokine and cytokine receptor gene expression in the adult mammalian heart.

BACKGROUND: Although previous studies have examined the effects of acute hemodynamic pressure overload on proinflammatory cytokine gene expression, the effects of sustained hemodynamic overloading have not been examined. METHODS AND RESULTS: Sustained hemodynamic pressure overloading was produced in mice by transverse constriction of the aorta. Proinflammatory cytokine and cytokine receptor gene expression were determined by ribonuclease protection assays (RPA) at 6 hours and at 3, 7, 14 and 35 days after banding. M-mode echocardiography was used to assess left ventricular structure and function at identical time points. RPA showed that tumor necrosis factor (TNF), interleukin (IL)-1beta, and IL-6 mRNA levels were maximal at 6 hours and returned to baseline levels within 72 hours. There was a significant increase in IL-1RII and IL-6Ralpha receptor mRNA levels after overloading but no significant increase in TNFR1, TNFR2, IL-1RI, or gp130 mRNA levels. The transient increase in expression of proinflammatory cytokine gene expression was not explained by changes in left ventricular loading conditions, left ventricular wall stress, desensitization of proinflammatory genes, or decreased nuclear factor-kappaB activation. It is interesting that transverse constriction of the aorta provoked an increase in the expression of tristetraprolin, a homeostatic zinc finger protein that is known to destabilize TNF mRNA. CONCLUSION: Sustained hemodynamic overloading provokes a transient increase in proinflammatory cytokine and cytokine receptor gene expression; however, the decrease in proinflammatory cytokine gene expression occurred in the absence of changes in loading conditions, suggesting that the expression of proinflammatory cytokines in the heart is regulated, at least in part, by load-dependent and load-independent mechanisms.

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.

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.

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.

Increased myocardial gene expression of tumor necrosis factor-alpha and nitric oxide synthase-2: a potential mechanism for depressed myocardial function in hibernating myocardium in humans.

BACKGROUND: Whether cardioinhibitory cytokines are elevated in regions of hibernating myocardium and account in part for the depression in resting function is currently not known. Methods and Results- Thirteen patients with stable ischemic ventricular dysfunction scheduled for bypass surgery underwent preoperative dobutamine echocardiography (DE) and intraoperative myocardial biopsies. The numbers of copies of mRNA for the negatively inotropic cytokines tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (NOS2) were quantified by reverse transcription-polymerase chain reaction. In normal segments, myocardial TNF-alpha was barely detectable (1.2+/-0.4 copies per 10(6) copies of beta-actin). A 13.7-fold increase in myocardial TNF-alpha was observed in dysfunctional segments with a biphasic response to DE (contractile reserve and ischemia) and was highest (45.5-fold) in segments with ischemia and without contractile reserve (P<0.001). A similar graded increase was seen for NOS2. Cytokine results were also similar if analysis was performed using recovery of function at 3 months as the index of viability. The change in serum TNF-alpha and nitrite levels from baseline to 3 months after surgery correlated inversely with both the change in ejection fraction and the number of DE viable segments (r=-0.92 to -0.93; P<0.001). CONCLUSIONS: TNF-alpha and NOS2 gene expression is regionally upregulated in hibernating myocardium to a level intermediate between that of normal regions and ischemic regions without contractile reserve. This, along with a decline in serum cytokine levels after revascularization proportional to the extent of myocardial viability, suggests a contributing role for cardioinhibitory cytokines in the observed depression of function seen in hibernating myocardium.

Myocardial proinflammatory cytokine expression and left ventricular remodeling in patients with chronic mitral regurgitation.

BACKGROUND: In an animal model, stretch was shown to induce myocardial tumor necrosis factor-alpha (TNF-alpha) expression. The purposes of this study were to determine whether the left ventricular (LV) volume overload that occurs in patients with chronic mitral regurgitation (MR) can induce myocardial and systemic TNF-alpha expression and whether there is a relationship between TNF-alpha expression and LV remodeling. METHODS AND RESULTS: Plasma TNF-alpha and its receptors were measured before mitral valve (MV) repair surgery in 26 MR patients and 23+/-12 months after MV repair surgery in 9 MR patients. Myocardial mRNA copies of TNF-alpha were determined in 11 MR and 10 donor hearts using quantitative RT-PCR. Compared with 15 control subjects, pre-MV repair plasma TNF-alpha (3.59+/-1.81 versus 2.03+/-1.02 pg/mL, P<0.005) and its receptor levels were elevated in MR patients. Myocardial TNF-alpha mRNA copies (corrected for beta-actin mRNA expression) in MR patients and donor hearts were 38.96+/-42.74x10(6) and 0.88+/-0.75x10(6), respectively (P=0.01). After MV surgery, there was a decrease in the plasma levels of TNF-alpha (2.79+/-1.14 versus 3.51+/-1.34 pg/mL, P=0.02) and its receptors. There was a correlation between myocardial TNF-alpha expression and preoperative LV end-diastolic and end-systolic volumes. Moreover, there was an inverse correlation between myocardial TNF-alpha expression and regression in LV end-diastolic (r=-0.76, P=0.007) and end-systolic (r=-0.73, P=0.01) volumes after MV surgery. CONCLUSIONS: TNF-alpha is expressed in the myocardium and plasma of MR patients. Correction of the LV volume overload with MV surgery results in reversal of TNF-alpha expression. There is a relationship between TNF-alpha expression and parameters of LV remodeling, suggesting that TNF-alpha may play a role in the pathogenesis of the LV remodeling that occurs in MR.

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.

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.

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.

Cardiac inflammation and innate immunity in septic shock: is there a role for toll-like receptors?

Our current understanding of the pathogenesis of sepsis suggests that bacteria as well as bacterial-derived products activate an uncontrolled network of host-derived mediators such as proinflammatory cytokines (ie, tumor necrosis factor [TNF] and interleukin [IL]-1beta), which can ultimately lead to cardiovascular collapse and death. Despite the potentially important role that TNF and IL-1beta may play in producing cardiac dysfunction in human septic shock, little is known with regard to the basic biochemical mechanism(s) by which bacterial pathogens induce their expression in the heart. A major advance in understanding the early events that are downstream from bacterial-mediated signaling has been the identification of Toll-like receptors (TLRs). TLR-mediated signaling is known to activate the transcription factor nuclear factor-kappaB and to upregulate TNF expression. It has recently been shown that the heart expresses TLRs, raising the possibility that these receptors may be responsible for mediating the deleterious effects of bacterial pathogens on cardiac function. In this review, we will discuss the emerging role for TLRs in the pathogenesis of the cardiovascular collapse that occurs during sepsis.

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 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.

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.