Tariq Hamid

Divergent Tumor Necrosis Factor Receptor–Related Remodeling Responses in Heart Failure Role of Nuclear Factor-κB and Inflammatory Activation

Background— Although preclinical data suggested that tumor necrosis factor-&agr; (TNF) neutralization in heart failure (HF) would be beneficial, clinical trials of TNF antagonists were paradoxically negative. We hypothesized that TNF induces opposing inflammatory and remodeling responses in HF that are TNF-receptor (TNFR) specific. Methods and Results— HF was induced in wild-type (WT), TNFR1−/−, and TNFR2−/− mice via coronary ligation. Compared with WT HF, 4-week postinfarction survival was significantly improved in both TNFR1−/− and TNFR2−/− HF. Compared with sham, WT HF hearts exhibited significant remodeling with robust activation of nuclear factor (NF)-&kgr;B, p38 mitogen-activated protein kinase, and JNK2 and upregulation of TNF, interleukin (IL)-1&bgr;, IL-6, and IL-10. Compared with WT HF, TNFR1−/− HF exhibited (1) improved remodeling, hypertrophy, and contractile function; (2) less apoptosis; and (3) diminished NF-&kgr;B, p38 mitogen-activated protein kinase, and JNK2 activation and cytokine expression. In contrast, TNFR2−/− HF showed exaggerated remodeling and hypertrophy, increased border zone fibrosis, augmented NF-&kgr;B and p38 mitogen-activated protein kinase activation, higher IL-1&bgr; and IL-6 gene expression, greater activated macrophages, and greater apoptosis. Oxidative stress and diastolic function were improved in both TNFR1−/−and TNFR2−/− HF. In H9c2 cardiomyocytes, sustained NF-&kgr;B activation was proapoptotic, an effect dependent on TNFR1 signaling, whereas TNFR2 overexpression attenuated TNF-induced NF-&kgr;B activation. Conclusions— TNFR1 and TNFR2 have disparate and opposing effects on remodeling, hypertrophy, NF-&kgr;B, inflammation, and apoptosis in HF: TNFR1 exacerbates, whereas TNFR2 ameliorates, these events. However, signaling through both receptors is required to induce diastolic dysfunction and oxidative stress. TNFR-specific effects in HF should be considered when therapeutic anti-TNF strategies are developed.

Cardioprotective and Antiapoptotic Effects of Heme Oxygenase-1 in the Failing Heart

Background— Heme oxygenase-1 (HO-1) is an inducible stress-response protein that imparts antioxidant and antiapoptotic effects. However, its pathophysiological role in cardiac remodeling and chronic heart failure (HF) is unknown. We hypothesized that induction of HO-1 in HF alleviates pathological remodeling. Methods and Results— Adult male nontransgenic and myocyte-restricted HO-1 transgenic mice underwent either sham operation or coronary ligation to induce HF. Four weeks after ligation, nontransgenic HF mice exhibited postinfarction left ventricular (LV) remodeling and dysfunction, hypertrophy, fibrosis, oxidative stress, apoptosis, and reduced capillary density, associated with a 2-fold increase in HO-1 expression in noninfarcted myocardium. Compared with nontransgenic mice, HO-1 transgenic HF mice exhibited significantly (P<0.05) improved postinfarction survival (94% versus 57%) and less LV dilatation (end-diastolic volume, 46±8 versus 85±32 &mgr;L), mechanical dysfunction (ejection fraction, 65±9% versus 49±16%), hypertrophy (LV/tibia length 4.4±0.4 versus 5.2±0.6 mg/mm), interstitial fibrosis (11.2±3.1% versus 18.5±3.5%), and oxidative stress (3-fold reduction in tissue malondialdehyde). Moreover, myocyte-specific HO-1 overexpression in HF promoted tissue neovascularization and ameliorated myocardial p53 expression (2-fold reduction) and apoptosis. In isolated mitochondria, mitochondrial permeability transition was inhibited by HO-1 in a carbon monoxide (CO)–dependent manner and was recapitulated by the CO donor tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). HO-1–derived CO also prevented H2O2-induced cardiomyocyte apoptosis and cell death. Finally, in vivo treatment with CORM-3 alleviated postinfarction LV remodeling, p53 expression, and apoptosis. Conclusions— HO-1 induction in the failing heart is an important cardioprotective adaptation that opposes pathological LV remodeling, and this effect is mediated, at least in part, by CO-dependent inhibition of mitochondrial permeability transition and apoptosis. Augmentation of HO-1 or its product, CO, may represent a novel therapeutic strategy for ameliorating HF.

H2S Protects Against Pressure Overload Induced Heart Failure via Upregulation of Endothelial Nitric Oxide Synthase (eNOS)

Background— Cystathionine &ggr;-lyase (CSE) produces H2S via enzymatic conversion of L-cysteine and plays a critical role in cardiovascular homeostasis. We investigated the effects of genetic modulation of CSE and exogenous H2S therapy in the setting of pressure overload–induced heart failure. Methods and Results— Transverse aortic constriction was performed in wild-type, CSE knockout, and cardiac-specific CSE transgenic mice. In addition, C57BL/6J or CSE knockout mice received a novel H2S donor (SG-1002). Mice were followed up for 12 weeks with echocardiography. We observed a >60% reduction in myocardial and circulating H2S levels after transverse aortic constriction. CSE knockout mice exhibited significantly greater cardiac dilatation and dysfunction than wild-type mice after transverse aortic constriction, and cardiac-specific CSE transgenic mice maintained cardiac structure and function after transverse aortic constriction. H2S therapy with SG-1002 resulted in cardioprotection during transverse aortic constriction via upregulation of the vascular endothelial growth factor–Akt–endothelial nitric oxide synthase–nitric oxide–cGMP pathway with preserved mitochondrial function, attenuated oxidative stress, and increased myocardial vascular density. Conclusions— Our results demonstrate that H2S levels are decreased in mice in the setting of heart failure. Moreover, CSE plays a critical role in the preservation of cardiac function in heart failure, and oral H2S therapy prevents the transition from compensated to decompensated heart failure in part via upregulation of endothelial nitric oxide synthase and increased nitric oxide bioavailability.

Cardiomyocyte NF-κB p65 promotes adverse remodelling, apoptosis, and endoplasmic reticulum stress in heart failure

AIMS the role of nuclear factor (NF)-κB in heart failure (HF) is not well defined. We sought to determine whether myocyte-localized NF-κB p65 activation in HF exacerbates post-infarction remodelling and promotes maladaptive endoplasmic reticulum (ER) stress. METHODS AND RESULTS non-transgenic (NTg) and transgenic (Tg) mice with myocyte-restricted overexpression of a phosphorylation-resistant inhibitor of κBα (IκBα(S32A,S36A)) underwent coronary ligation (to induce HF) or sham operation. Over 4 weeks, the remote myocardium of ligated hearts exhibited robust NF-κB activation that was almost exclusively p65 beyond 24 h. Compared with sham at 4 weeks, NTg HF hearts were dilated and dysfunctional, and exhibited hypertrophy, fibrosis, up-regulation of inflammatory cytokines, increased apoptosis, down-regulation of ER protein chaperones, and up-regulation of the ER stress-activated pro-apoptotic factor CHOP. Compared with NTg HF, Tg-IκBα(S32A,S36A) HF mice exhibited: (i) improved survival, chamber remodelling, systolic function, and pulmonary congestion, (ii) markedly diminished NF-κB p65 activation, cytokine expression, and fibrosis, and (iii) a three-fold reduction in apoptosis. Moreover, Tg-IκBα(S32A,S36A) HF hearts exhibited maintained expression of ER chaperones and CHOP when compared with sham. In cardiomyocytes, NF-κB activation was required for ER stress-mediated apoptosis, whereas abrogation of myocyte NF-κB shifted the ER stress response to one of adaptation and survival. CONCLUSION persistent myocyte NF-κB p65 activation in HF exacerbates cardiac remodelling by imparting pro-inflammatory, pro-fibrotic, and pro-apoptotic effects. p65 modulation of cell death in HF may occur in part from NF-κB-mediated transformation of the ER stress response from one of adaptation to one of apoptosis.

Remodeling of the Mononuclear Phagocyte Network Underlies Chronic Inflammation and Disease Progression in Heart Failure: Critical Importance of the Cardiosplenic Axis

Rationale: The role of mononuclear phagocytes in chronic heart failure (HF) is unknown. Objective: Our aim was to delineate monocyte, macrophage, and dendritic cell trafficking in HF and define the contribution of the spleen to cardiac remodeling. Methods and Results: We evaluated C57Bl/6 mice with chronic HF 8 weeks after coronary ligation. As compared with sham-operated controls, HF mice exhibited: (1) increased proinflammatory CD11b+F4/80+CD206− macrophages and CD11b+F4/80+Gr-1hi monocytes in the heart and peripheral blood, respectively, and reduced CD11b+F4/80+Gr-1hi monocytes in the spleen; (2) significantly increased CD11c+B220− classical dendritic cells and CD11c+/lowB220+ plasmacytoid dendritic cells in both the heart and spleen, and increased classic dendritic cells and plasmacytoid dendritic cells in peripheral blood and bone marrow, respectively; (3) increased CD4+ helper and CD8+ cytotoxic T-cells in the spleen; and (4) profound splenic remodeling with abundant white pulp follicles, markedly increased size of the marginal zone and germinal centers, and increased expression of alarmins. Splenectomy in mice with established HF reversed pathological cardiac remodeling and inflammation. Splenocytes adoptively transferred from mice with HF, but not from sham-operated mice, homed to the heart and induced long-term left ventricular dilatation, dysfunction, and fibrosis in naive recipients. Recipient mice also exhibited monocyte activation and splenic remodeling similar to HF mice. Conclusions: Activation of mononuclear phagocytes is central to the progression of cardiac remodeling in HF, and heightened antigen processing in the spleen plays a critical role in this process. Splenocytes (presumably splenic monocytes and dendritic cells) promote immune-mediated injurious responses in the failing heart and retain this memory on adoptive transfer.

PTTG/securin activates expression of p53 and modulates its function

BackgroundPituitary tumor transforming gene (PTTG) is a novel oncogene that is expressed abundantly in most tumors. Overexpression of PTTG induces cellular transformation and promotes tumor formation in nude mice. PTTG has been implicated in various cellular processes including sister chromatid separation during cell division as well as induction of apoptosis through p53-dependent and p53-independent mechanisms. The relationship between PTTG and p53 remains unclear, however.ResultsHere we report the effects of overexpression of PTTG on the expression and function of p53. Our results indicate that overexpression of PTTG regulates the expression of the p53 gene at both the transcriptional and translational levels and that this ability of PTTG to activate the expression of p53 gene is dependent upon the p53 status of the cell. Deletion analysis of the p53 gene promoter revealed that only a small region of the p53 gene promoter is required for its activation by PTTG and further indicated that the activation of p53 gene by PTTG is an indirect effect that is mediated through the regulation of the expression of c-myc, which then interacts with the p53 gene promoter. Our results also indicate that overexpression of PTTG stimulates expression of the Bax gene, one of the known downstream targets of p53, and induces apoptosis in a human embryonic kidney cell line (HEK293). This stimulation of bax expression by PTTG is indirect and is mediated through modulation of p53 gene expression.ConclusionsOverexpression of PTTG activates the expression of p53 and modulates its function, with this action of PTTG being mediated through the regulation of c-myc expression. PTTG also up-regulates the activity of the bax promoter and increases the expression of bax through modulation of p53 expression.

Ectopic expression of PTTG1/securin promotes tumorigenesis in human embryonic kidney cells

BackgroundPituitary tumor transforming gene1 (PTTG1) is a novel oncogene that is expressed in most tumors. It encodes a protein that is primarily involved in the regulation of sister chromatid separation during cell division. The oncogenic potential of PTTG1 has been well characterized in the mouse, particularly mouse fibroblast (NIH3T3) cells, in which it induces cell proliferation, promotes tumor formation and angiogenesis. Human tumorigenesis is a complex and a multistep process often requiring concordant expression of a number of genes. Also due to differences between rodent and human cell biology it is difficult to extrapolate results from mouse models to humans. To determine if PTTG1 functions similarly as an oncogene in humans, we have characterized its effects on human embryonic kidney (HEK293) cells.ResultsWe report that introduction of human PTTG1 into HEK293 cells through transfection with PTTG1 cDNA resulted in increased cell proliferation, anchorage-independent growth in soft agar, and formation of tumors after subcutaneous injection of nu/nu mice. Pathologic analysis revealed that these tumors were poorly differentiated. Both analysis of HEK293 cells transiently transfected with PTTG1 cDNA and analysis of tumors developed on injection of HEK293 cells that had been stably transfected with PTTG1 cDNA indicated significantly higher levels of secretion and expression of bFGF, VEGF and IL-8 compared to HEK293 cells transfected with pcDNA3.1 vector or uninvolved tissues collected from the mice. Mutation of the proline-rich motifs at the C-terminal of PTTG1 abolished its oncogenic properties. Mice injected with this mutated PTTG1 either did not form tumors or formed very small tumors. Taken together our results suggest that PTTG1 is a human oncogene that possesses the ability to promote tumorigenesis in human cells at least in part through the regulation of expression or secretion of bFGF, VEGF and IL-8.ConclusionsOur results demonstrate that PTTG1 is a potent human oncogene and has the ability to induce cellular transformation of human cells. Overexpression of PTTG1 in HEK293 cells leads to an increase in the secretion and expression of bFGF, VEGF and IL-8. Mutation of C-terminal proline-rich motifs abrogates the oncogenic function of PTTG1. To our knowledge, this is the first study demonstrating the importance of PTTG1 in human tumorigenesis.

O-GlcNAc signaling attenuates ER stress-induced cardiomyocyte death

We previously demonstrated that the O-linked beta-N-acetylglucosamine (O-GlcNAc) posttranslational modification confers cardioprotection at least partially through mitochondrial-dependent mechanisms, but it remained unclear if O-GlcNAc signaling interfered with other mechanisms of cell death. Because ischemia/hypoxia causes endoplasmic reticulum (ER) stress, we ascertained whether O-GlcNAc signaling could attenuate ER stress-induced cell death per se. Before induction of ER stress (with tunicamycin or brefeldin A), we adenovirally overexpressed O-GlcNAc transferase (AdOGT) or pharmacologically inhibited O-GlcNAcase [via O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate] to augment O-GlcNAc levels or adenovirally overexpressed O-GlcNAcase to reduce O-GlcNAc levels. AdOGT significantly (P < 0.05) attenuated the activation of the maladaptive arm of the unfolded protein response [according to C/EBP homologous protein (CHOP) activation] and cardiomyocyte death (reflected by percent propidium iodide positivity). Moreover, pharmacological inhibition of O-GlcNAcase significantly (P < 0.05) mitigated ER stress-induced CHOP activation and cardiac myocyte death. Interestingly, overexpression of GCA did not alter ER stress markers but exacerbated brefeldin A-induced cardiomyocyte death. We conclude that enhanced O-GlcNAc signaling represents a partially proadaptive response to reduce ER stress-induced cell death. These results provide new insights into a possible interaction between O-GlcNAc signaling and ER stress and may partially explain a mechanism of O-GlcNAc-mediated cardioprotection.

O-GlcNAc signaling is essential for NFAT-mediated transcriptional reprogramming during cardiomyocyte hypertrophy

The regulation of cardiomyocyte hypertrophy is a complex interplay among many known and unknown processes. One specific pathway involves the phosphatase calcineurin, which regulates nuclear translocation of the essential cardiac hypertrophy transcription factor, nuclear factor of activated T-cells (NFAT). Although metabolic dysregulation is frequently described during cardiac hypertrophy, limited insights exist regarding various accessory pathways. One metabolically derived signal, beta-O-linked N-acetylglucosamine (O-GlcNAc), has emerged as a highly dynamic posttranslational modification of serine and threonine residues regulating physiological and stress processes. Given the metabolic dysregulation during hypertrophy, we hypothesized that NFAT activation is dependent on O-GlcNAc signaling. Pressure overload-induced hypertrophy (via transverse aortic constriction) in mice or treatment of neonatal rat cardiac myocytes with phenylephrine significantly enhanced global O-GlcNAc signaling. NFAT-luciferase reporter activity revealed O-GlcNAc-dependent NFAT activation during hypertrophy. Reversal of enhanced O-GlcNAc signaling blunted cardiomyocyte NFAT-induced changes during hypertrophy. Taken together, these results demonstrate a critical role of O-GlcNAc signaling in NFAT activation during hypertrophy and provide evidence that O-GlcNAc signaling is coordinated with the onset and progression of cardiac hypertrophy. This represents a potentially significant and novel mechanism of cardiac hypertrophy, which may be of particular interest in future in vivo studies of hypertrophy.

Chronic oral exposure to the aldehyde pollutant acrolein induces dilated cardiomyopathy

Environmental triggers of dilated cardiomyopathy are poorly understood. Acute exposure to acrolein, a ubiquitous aldehyde pollutant, impairs cardiac function and cardioprotective responses in mice. Here, we tested the hypothesis that chronic oral exposure to acrolein induces inflammation and cardiomyopathy. C57BL/6 mice were gavage-fed acrolein (1 mg/kg) or water (vehicle) daily for 48 days. The dose was chosen based on estimates of human daily unsaturated aldehyde consumption. Compared with vehicle-fed mice, acrolein-fed mice exhibited significant (P < 0.05) left ventricular (LV) dilatation (LV end-diastolic volume 36 ± 8 vs. 17 ± 5 μl), contractile dysfunction (dP/dt(max) 4,697 ± 1,498 vs. 7,016 ± 1,757 mmHg/s), and impaired relaxation (tau 15.4 ± 4.3 vs. 10.4 ± 2.2 ms). Histological and biochemical evaluation revealed myocardial oxidative stress (membrane-localized protein-4-hydroxy-trans-2-nonenal adducts) and nitrative stress (increased protein-nitrotyrosine) and varying degrees of plasma and myocardial protein-acrolein adduct formation indicative of physical translocation of ingested acrolein to the heart. Acrolein also induced myocyte hypertrophy (~2.2-fold increased myocyte area, P < 0.05), increased apoptosis (~7.5-fold), and disrupted endothelial nitric oxide synthase in the heart. DNA binding studies, immunohistochemistry, and PCR revealed significant (P < 0.05) activation of nuclear factor-κB in acrolein-exposed hearts, along with upregulated gene expression of proinflammatory cytokines tumor necrosis factor-α and interleukin-1β. Long-term oral exposure to acrolein, at an amount within the range of human unsaturated aldehyde intake, induces a phenotype of dilated cardiomyopathy in the mouse. Human exposure to acrolein may have analogous effects and raise consideration of an environmental, aldehyde-mediated basis for heart failure.