REDD1 attenuates cardiac hypertrophy via enhancing autophagy

Cardiac hypertrophy is a major risk factor of cardiovascular morbidity and mortality. Autophagy is established to be involved in regulating cardiac hypertrophy. REDD1, a stress-responsive protein, is proved to contribute in autophagy induction. However, the role of REDD1 in cardiac hypertrophy remains unknown. Our study demonstrated that REDD1 knockdown by RNAi exacerbated phenylephrine (PE)-induced cardiac hypertrophy, manifested by increased hypertrophic markers such as ANP and cell surface area. In addition, we discovered that ERK1/2 signaling pathway was involved in the effect of REDD1 on hypertrophy. Moreover, our study showed that REDD1 knockdown impaired autophagy in hypertrophied cardiomyocytes. mTOR, a signaling molecule governing autophagy induction, was activated by the knockdown of REDD1 under PE stress. Importantly, the pro-hypertrophic effect of REDD1 knockdown was significantly reversed by the autophagy enhancer rapamycin. Taken together, we firstly prove that REDD1 is essential for inhibiting cardiac hypertrophy by enhancing autophagy.     

The chemical chaperone 4-phenylbutyric acid attenuates pressure-overload cardiac hypertrophy by alleviating endoplasmic reticulum stress

Evidence has shown that endoplasmic reticulum stress (ERS) is associated with the pathogenesis of cardiac hypertrophy. The aim of this study was to investigate whether direct alleviation of ER stress by 4-phenylbutyric acid (PBA), a known chemical chaperone drug, could attenuate pressure-overload cardiac hypertrophy in mice. The effects of orally administered PBA (100mg/kg body weight daily for a week) were examined using mice undergoing transverse aortic constriction (TAC-mice), an animal model to produce pressure overload. TAC application for 1 week led to a 1.8-fold increase in the ratio of the heart weight over body weight (HW/BW) and up-regulation of the hypertrophy markers ANF and BNF accompanied by up-regulation of ERS markers (GRP78, p-PERK, and p-elF2α). The oral administration of PBA to the TAC-mice reduced hypertrophy (19%) and severely downregulated the fibrosis-related genes (transforming growth factor-β1, phospho-smad2, and pro-collagen isoforms). We conclude that ERS is induced as a consequence of remodeling during pathological hypertrophy and that PBA may help to relieve ERS and play a protective role against cardiac hypertrophy and possibly heart failure. We suggest PBA as a novel therapeutic agent for cardiac hypertrophy and fibrosis.     

Mononuclear cells enhance prostaglandin E2 production of polymorphonuclear leukocytes via tumor necrosis factor alpha

To clarify the interactions between mononuclear cells and polymorphonuclear leukocytes, and to identify the cytokine(s) that mediate the interaction, the effects of a culture supernatant of LPS-stimulated mononuclear cells on production of arachidonic acid metabolites of polymorphonuclear cells were studied. The culture supernatant of LPS-stimulated mononuclear cells increased production of prostaglandin E2 of polymorphonuclear cells. TNF alpha, but not IL-1, IL-2, IL-6, or IFN gamma, enhanced the prostaglandin E2 production when added in vitro. Additionally, an anti-rTNF alpha monoclonal antibody inhibited the stimulating activity of the culture supernatants. TNF alpha, produced by mononuclear cells, appears to play an important role in the development of inflammation, such as rheumatoid arthritis, by enhancing the arachidonic acid metabolism of the polymorphonuclear cells.     

Matrix metalloproteinase-2 contributes to tumor necrosis factor alpha induced apoptosis in cultured rat cardiac myocytes

Tumor necrosis factor alpha (TNFalpha) is associated with a higher risk of cardiovascular disease. Matrix metalloproteinase-2 (MMP-2) has been implicated in the pathophysiology of ischemic heart disease. However, the role of interactions between MMP-2 and TNFalpha, associated with cardiac apoptosis, is unknown. We hypothesized that MMP-2 will contribute to TNFalpha-induced myocardial apoptosis. After treatment with TNFalpha (1-20 ng/ml) for 24 h, or with TNFalpha (10 ng/ml) for 0, 6, 12, 24, or 48 h, MMP-2 activity, percent of TUNEL-positive myocytes, and DNA fragmentation dose, and time-dependently increased compared to control. However, TNFalpha blockade (neutralizing antibodies against human TNFalpha, 25 microg/ml) significantly reduced the activity of MMP-2 and markers of apoptosis induced by TNFalpha. Interestingly, MMP-2 antibody (30 microg/ml), or the MMP-2 inhibitors Doxycycline (Dox, 1-50 micromol/l) or GM6001 (GM, 10 micromol/l), prior to TNFalpha insult, decreased myocardial MMP-2 activity and reduced the percent of TUNEL-positive myocytes and DNA fragmentation. Moreover, MMP-2 inhibition reduced Bax expression and caspase3 activity, as well as increasing Bcl2 expression. MMP-2 inhibition was associated with decreased cardiac MMP-2 activity and decreased myocardial apoptosis induced by TNFalpha. These results suggest that MMP-2 contributes to TNFalpha-induced apoptosis in cultured rat cardiac myocytes.     

Absence of host tumor necrosis factor receptor 1 attenuates manifestations of idiopathic pneumonia syndrome

The interaction of TNF-alpha with TNF receptor 1 (TNFR1) activates several signal transduction pathways that lead to apoptosis or NF-kappa B-dependent inflammation and immunity. We hypothesized that host TNFR1 expression contributes to noninfectious lung injury and inflammation commonly observed after bone marrow transplantation (BMT), termed idiopathic pneumonia syndrome (IPS). C57BL/6 TNFR1-sufficient (TNFR1(+/+)) and -deficient (TNFR1(-/-)) mice were total body irradiated with or without cyclophosphamide conditioning and were given bone marrow plus IPS-inducing donor spleen T cells from B10.BR wild-type mice. TNFR1(-/-) recipient mice exhibited improved early post-BMT survival associated with decreased permeability edema. In addition, the low lung compliance measured in anesthetized, ventilated TNFR1(+/+) mice on day 7 after BMT was restored to baseline during TNFR1 deficiency. Importantly, bronchoalveolar lavage fluid (BALF) inflammatory cells from TNFR1(-/-) vs. TNFR1(+/+) mice generated less nitric oxide (.NO) and nitrating species and exhibited suppressed programmed cell death as assessed using flow cytometry. However, cellular infiltration and levels of proinflammatory cytokines and chemokines were generally higher in BALF collected on day 7 after BMT from TNFR1(-/-) compared with TNFR1(+/+) recipient mice. Our results support a major role of host TNFR1 in regulation of .NO production and lung dysfunction after allogeneic BMT.     

Increased natriuretic peptide receptor A and C gene expression in rats with pressure-overload cardiac hypertrophy

Both atrial (ANP) and brain (BNP) natriuretic peptide affect development of cardiac hypertrophy and fibrosis via binding to natriuretic peptide receptor (NPR)-A in the heart. A putative clearance receptor, NPR-C, is believed to regulate cardiac levels of ANP and BNP. The renin-angiotensin system also affects cardiac hypertrophy and fibrosis. In this study we examined the expression of genes for the NPRs in rats with pressure-overload cardiac hypertrophy. The ANG II type 1 receptor was blocked with losartan (10 mg.kg(-1).day(-1)) to investigate a possible role of the renin-angiotensin system in regulation of natriuretic peptide and NPR gene expression. The ascending aorta was banded in 84 rats during Hypnorm/Dormicum-isoflurane anesthesia; after 4 wk the rats were randomized to treatment with losartan or placebo. The left ventricle of the heart was removed 1, 2, or 4 wk later. Aortic banding increased left ventricular expression of NPR-A and NPR-C mRNA by 110% (P < 0.001) and 520% (P < 0.01), respectively, after 8 wk; as expected, it also increased the expression of ANP and BNP mRNAs. Losartan induced a slight reduction of left ventricular weight but did not affect the expression of mRNAs for the natriuretic peptides or their receptors. Although increased gene expression does not necessarily convey a higher concentration of the protein, the data suggest that pressure overload is accompanied by upregulation of not only ANP and BNP but also their receptors NPR-A and NPR-C in the left ventricle.     

Renin-angiotensin system and sympathetic nervous system in cardiac pressure-overload hypertrophy

Angiotensin II and norepinephrine (NE) have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy. The purpose of this study was to determine the temporal sequence for activation of the renin-angiotensin and sympathetic nervous systems in the rat after 3-60 days of pressure overload induced by aortic constriction. Initially on pressure overload, there was transient activation of the systemic renin-angiotensin system coinciding with the appearance of left ventricular hypertrophy (day 3). At day 10, there was a marked increase in AT(1) receptor density in the left ventricle, increased plasma NE concentration, and elevated cardiac epinephrine content. Moreover, the inotropic response to isoproterenol was reduced in the isolated, perfused heart at 10 days of pressure overload. The affinity of the beta(2)-adrenergic receptor in the left ventricle was decreased at 60 days. Despite these alterations, there was no decline in resting left ventricular function, beta-adrenergic receptor density, or the relative distribution of beta(1)- and beta(2)-receptor sites in the left ventricle over 60 days of pressure overload. Thus activation of the renin-angiotensin system is an early response to pressure overload and may contribute to the initial development of cardiac hypertrophy and sympathetic activation in the compensated heart.     

Novel muteins of human tumor necrosis factor alpha

For chemical synthesis of a gene coding for human tumor necrosis factor alpha (TNF-alpha), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 bp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into a vector (pKK223-3) for high expression of active TNF-alpha in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-alpha were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-alpha. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-beta. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-alpha. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.     

Tamoxifen protects against acute tumor necrosis factor alpha-induced cardiac injury via improving mitochondrial functions

Tamoxifen is the most commonly used antiestrogen for the treatment of breast cancer. Several clinical trials demonstrate that tamoxifen reduces the risk of heart disease and osteoporosis. However, the mechanism by which tamoxifen causes cardioprotection is unclear. Because increased levels of tumor necrosis factor alpha (TNFalpha) in tissue and/or plasma have been observed in virtually all forms of cardiac injury, we investigated whether tamoxifen prevents cardiac injury in a murine model of acute TNFalpha challenge. Five- to six-week-old female mice were injected (ip) with tamoxifen at 0.25 mg/kg daily for 3 or 7 days before receiving an injection of TNFalpha. Ultrastructural examination of cardiac tissues revealed remarkable protection against TNFalpha-induced mitochondrial damage in tamoxifen pretreated mice. Tamoxifen treatment significantly improved the mitochondrial respiratory function and enhanced superoxide-scavenging activity of mitochondria. These findings reveal a novel mitochondria-mediated mechanism by which tamoxifen exerts its cardiac protection effect against acute TNFalpha-induced heart injury.     

Allelic variation of the type 2 tumor necrosis factor receptor gene

The nucleotide sequence data reported in this paper have been submitted to the EMBL Data library and have been assigned the accession number X76401.