Tumor necrosis factor induced oxidative stress in the central nervous system contributes to sympathoexcitation in heart failure

Despite advanced therapeutic strategies for post-myocardial infarction (MI) patients, many ultimately develop congestive heart failure (CHF), rendering the disease a major cause of death in the United States. MI is associated with an acute increase in sympathetic nervous system activity, becoming persistent in CHF patients. Increased pro-inflammatory cytokines (PICs) following MI are implicated in the pathogenesis of CHF. The increase in tumor necrosis factor (TNF), a primary PIC, correlates closely with heart disease severity. Moreover, central PIC production increases post-MI, and can affect the brain’s cardiovascular regulatory regions that control sympathoexcitation. Therefore, understanding how PICs modulate sympathoexcitation is important for development of new therapeutics. Recent studies underscore the importance of central NADPH oxidases in the pathogenesis of hypertension. However, the role of central NADPH oxidase-induced reactive oxygen species (ROS) production in the development of CHF remains limited. In this dissertation, the hypothesis that central PICs induce ROS production and modulate sympathoexcitatory neurons of the paraventricular nucleus (PVN) is explored through an array of selective animal models combined with novel technologies for sympathoexcitation and cardiovascular function assessment. The effect of the TNF blocker, pentoxyfylline, was investigated on the expression of the catalytic subunits of NADPH oxidase (Noxs) in the PVN neurons and on the sympathetic activity in CHF rats. Additionally, effects of TNF inhibition on central nitric oxide were explored, as this ROS restrains sympathoexcitation. More specifically, central TNF was inhibited to understand the interaction between superoxide and nitric oxide in the PVN neurons during CHF. TNF knock-out mice were also used to study the effect of TNF on volume overload associated with CHF. Finally, to understand the role of peripheral TNF on the PVN’s sympathoexcitatory neurons, and to exclude the effects of neurohormones in CHF, human recombinant TNF was injected 5-days systemically to achieve the levels observed following MI in conjunction with ROS and angiotensin II type-1 receptor blockers. These studies provide new evidence that TNF induces oxidative stress in the PVN through an AT1R mediated mechanism in CHF, and offers new insight into the sympathoexcitatory mechanisms in the brain possibly involved in the pathogenesis of CHF

Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans

Central TNF inhibition results in attenuated neurohumoral excitation in heart failure: a role for superoxide and nitric oxide

This study examined the effect of central tumor necrosis factor-alpha (TNF) blockade on the imbalance between nitric oxide and superoxide production in the paraventricular nucleus (PVN) and ventrolateral medulla (VLM), key autonomic regulators, and their contribution to enhanced sympathetic drive in mice with congestive heart failure (CHF). We also used a TNF gene knockout (KO) mouse model to study the involvement of TNF in body fluid homeostasis and sympathoexcitation in CHF. After implantation of intracerebroventricular (ICV) cannulae, myocardial infarction (MI) was induced in wild-type (WT) and KO mice by coronary artery ligation. Osmotic mini-pumps were implanted into one set of WT + MI/Sham mice for continuous ICV infusion of Etanercept (ETN), a TNF receptor fusion protein, or vehicle (VEH). Gene expressions of neuronal nitric oxide synthase (NOS) and angiotensin receptor-type 2 were reduced, while those of inducible NOS, Nox2 homologs, superoxide, peroxynitrite and angiotensin receptor-type 1 were elevated in the brainstem and hypothalamus of MI + VEH. Plasma norepinephrine levels and the number of Fos-positive neurons were also increased in the PVN and VLM in MI + VEH. MI + ETN and KO + MI mice exhibited reduced oxidative stress, reduced sympathoexcitation and an improved cardiac function. These changes in WT + MI were associated with increased sodium and fluid retention. These results indicate that elevated TNF in these autonomic regulatory regions of the brain alter the production of superoxide and nitric oxide, contributing to fluid imbalance and sympathoexcitation in CHF

Cytokine blockade attenuates sympathoexcitation in heart failure: cross-talk between nNOS, AT-1R and cytokines in the hypothalamic paraventricular nucleus

OBJECTIVE: To investigate evidence for the interplay between cytokines, angiotensin II and nNOS in the paraventricular nucleus (PVN), for regulating sympathetic outflow in a rat model of CHF. METHODS AND RESULTS: Heart failure was induced in Sprague-Dawley rats by coronary artery ligation. One group of rats was treated with pentoxifylline (PTX, 30 mg/kg IP), a cytokine blocker, or vehicle, for 5 weeks. Another group of rats was pre-treated with PTX before coronary ligation to study prior cytokine blocking effect on survival. Both groups were combined in the analysis. Echocardiography demonstrated an increase in LV end-diastolic pressure and Tei index after 5 weeks in CHF rats. ELISA revealed a significant increase in plasma TNF-alpha and IL-1beta in CHF rats. Inducible NOS (iNOS) and angiotensin receptor-type 1 (AT-1R) mRNA expressions were increased, while neuronal NOS (nNOS) was decreased in the PVN of CHF rats; these changes were reversed by PTX. PTX treatment also decreased plasma norepinephrine and epinephrine levels and improved baroreflex control of renal sympathoexcitation in CHF rats. Immunohistochemistry revealed elevated 3-nitrotyrosine formation in the heart and the PVN of CHF rats, but not in PTX treated rats. CONCLUSION: PTX decreased both peripheral and central cytokine expression, alleviated nitric oxide dysregulation, and inhibited the formation of peroxynitrite in the PVN resulting in decreased sympathoexcitation in CHF rats

TNF-alpha blockade decreases oxidative stress in the paraventricular nucleus and attenuates sympathoexcitation in heart failure rats

Oxidative stress plays an important role in the pathophysiology of cardiovascular disease. Recent evidence suggests that cytokines induce oxidative stress and contribute to cardiac dysfunction. In this study, we investigated whether increased circulating and tissue levels of tumor necrosis factor (TNF)-alpha in congestive heart failure (CHF) modulate the expression of NAD(P)H oxidase subunits, Nox2 and its isoforms, in the paraventricular nucleus (PVN) of the hypothalamus and contribute to exaggerated sympathetic drive in CHF. Heart failure was induced in Sprague-Dawly rats by coronary artery ligation and was confirmed using echocardiography. Pentoxifylline (PTX) was used to block the production of cytokines for a period of 5 wk. CHF induced a significant increase in the production of reactive oxygen species (ROS) in the left ventricle (LV) and in the PVN. The mRNA and protein expression of TNF-alpha, Nox1, Nox2, and Nox4 was significantly increased in the LV and PVN of CHF rats. CHF also decreased ejection fraction, increased Tei index, and increased circulating catecholamines (epinephrine and norepinephrine) and renal sympathetic activity (RSNA). In contrast, treatment with PTX in CHF rats completely blocked oxidative stress and decreased the production of TNF-alpha and Nox2 isoforms both in the LV and PVN. PTX treatment also decreased catecholamines and RSNA and prevented further decrease in cardiac function. In summary, TNF-alpha blockade attenuates ROS and sympathoexcitation in CHF. This study unveils new mechanisms by which cytokines play a role in the pathogenesis of CHF, thus underscoring the importance of targeting cytokines in heart failure

HDAC inhibition attenuates inflammatory, hypertrophic, and hypertensive responses in spontaneously hypertensive rats

Reactive oxygen species and proinflammatory cytokines contribute to cardiovascular diseases. Inhibition of downstream transcription factors and gene modifiers of these components are key mediators of hypertensive response. Histone acetylases/deacetylases can modulate the gene expression of these hypertrophic and hypertensive components. Therefore, we hypothesized that long-term inhibition of histone deacetylase with valproic acid might attenuate hypertrophic and hypertensive responses by modulating reactive oxygen species and proinflammatory cytokines in SHR rats. Seven-week-old SHR and WKY rats were used in this study. Following baseline blood pressure measurement, rats were administered valproic acid in drinking water (0.71% wt/vol) or vehicle, with pressure measured weekly thereafter. Another set of rats were treated with hydralazine (25 mg/kg per day orally) to determine the pressure-independent effects of HDAC inhibition on hypertension. Following 20 weeks of treatment, heart function was measured using echocardiography, rats were euthanized, and heart tissue was collected for measurement of total reactive oxygen species, as well as proinflammatory cytokine, cardiac hypertrophic, and oxidative stress gene and protein expressions. Blood pressure, proinflammatory cytokines, hypertrophic markers, and reactive oxygen species were increased in SHR versus WKY rats. These changes were decreased in valproic acid-treated SHR rats, whereas hydralazine treatment only reduced blood pressure. These data indicate that long-term histone deacetylase inhibition, independent of the blood pressure response, reduces hypertrophic, proinflammatory, and hypertensive responses by decreasing reactive oxygen species and angiotensin II type1 receptor expression in the heart, demonstrating the importance of uncontrolled histone deacetylase activity in hypertension

Chronic NF-κB blockade reduces cytosolic and mitochondrial oxidative stress and attenuates renal injury and hypertension in SHR

Nuclear factor-κB (NF-κB) plays an important role in hypertensive renal injury; however, its roles in perpetuating mitochondrial oxidative stress and renal dysfunction remain unclear. In this study, we assessed the effects of chronic NF-κB blockade with pyrrolidine dithiocarbamate (PDTC) on renal dysfunction and mitochondrial redox status in spontaneously hypertensive rats (SHR). PDTC (150 mg·kg body wt−1·day−1) or vehicle was administered orally to 8-wk-old SHR and their respective controls for 15 wk. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography at the start of and at every third week throughout the study. After 15 wk of treatment, anesthetized rats underwent acute renal experiments to determine renal blood flow and glomerular filtration rate using PAH and inulin clearance techniques, respectively. Following renal experiments, kidneys were excised from killed rats, and cortical mitochondria were isolated for reactive oxygen species (ROS) measurements using electron paramagnetic resonance. Tissue mRNA and protein levels of NF-κB and oxidative stress genes were determined using real-time PCR and immunofluorescence or Western blotting, respectively. PDTC treatment partially attenuated the increase in SBP (196.4 ± 9.76 vs. 151.4 ± 2.12; P < 0.05) and normalized renal hemodynamic and excretory parameters and ATP production rates in SHR. PDTC treatment also attenuated the higher levels of cytosolic and mitochondrial ROS generation and tissue mRNA and protein expression levels of NF-κB and oxidative stress genes in SHR without any comparable responses in control rats. These findings suggest that NF-κB activation by ROS induces the cytosolic and mitochondrial oxidative stress and tissue injury that contribute to renal dysfunction observed in SHR

Additional file 2: Movie S1. of Muscleblind-like 1 is required for normal heart valve development in vivo

Imaging of the AVC cushions in a representative wild type heart at E10.5 by OCT. Optical sections through a representative E10.5 wild type heart are shown. The AVC cushions are highlighted in purple, and a three-dimensional reconstruction of the AVC cushions is shown. A still image of this 3D reconstruction is shown in Fig. 3h, but has been rotated and scaled to match the orientation and magnification of the Mbnl1 ∆E3/∆E3 image in Fig. 3i. (MOV 2333 kb