Cardiac hypertrophy and altered beta-adrenergic signaling in transgenic mice that express the amino terminus of beta-ARK1

The G protein-coupled receptor (GPCR) kinase beta-adrenergic receptor (beta-AR) kinase-1 (beta-ARK1) is elevated during heart failure; however, its role is not fully understood. Beta-ARK1 contains several domains that are capable of protein-protein interactions that may play critical roles in the regulation of GPCR signaling. In this study, we developed a novel line of transgenic mice that express an amino-terminal peptide of beta-ARK1 that is comprised of amino acid residues 50-145 (beta-ARKnt) in the heart to determine whether this domain has any functional significance in vivo. Surprisingly, the beta-ARKnt transgenic mice presented with cardiac hypertrophy. Our data suggest that the phenotype was driven via an enhanced beta-AR system, as beta-ARKnt mice had elevated cardiac beta-AR density. Moreover, administration of a beta-AR antagonist reversed hypertrophy in these mice. Interestingly, signaling through the beta-AR in response to agonist stimulation was not enhanced in these mice. Thus the amino terminus of beta-ARK1 appears to be critical for normal beta-AR regulation in vivo, which further supports the hypothesis that beta-ARK1 plays a key role in normal and compromised cardiac GPCR signaling.     

Histone deacetylase-3 interacts with ataxin-7 and is altered in a spinocerebellar ataxia type 7 mouse model

Spinocerebellar ataxia type 7 (SCA7) is caused by a toxic polyglutamine (polyQ) expansion in the N-terminus of the protein ataxin-7. Ataxin-7 has a known function in the histone acetylase complex, Spt/Ada/Gcn5 acetylase (STAGA) chromatin-remodeling complex. We hypothesized that some histone deacetylase (HDAC) family members would impact the posttranslational modification of normal and expanded ataxin-7 and possibly modulate ataxin-7 function or neurotoxicity associated with the polyQ expansion. Interestingly, when we coexpressed each HDAC family member in the presence of ataxin-7 we found that HDAC3 increased the posttranslational modification of normal and expanded ataxin-7. Specifically, HDAC3 stabilized ataxin-7 and increased modification of the protein. Further, HDAC3 physically interacts with ataxin-7. The physical interaction of HDAC3 with normal and polyQ-expanded ataxin-7 affects the toxicity in a polyQ-dependent manner. We detect robust HDAC3 expression in neurons and glia in the cerebellum and an increase in the levels of HDAC3 in SCA7 mice. Consistent with this we found altered lysine acetylation levels and deacetylase activity in the brains of SCA7 transgenic mice. This study implicates HDAC3 and ataxin-7 interaction as a target for therapeutic intervention in SCA7, adding to a growing list of neurodegenerative diseases that may be treated by HDAC inhibitors.     

Cardiac phospholipidome is altered during ischemia and reperfusion in an ex vivo rat model

Acute myocardial infarction (AMI) is the leading cause of death, morbidity, and health costs worldwide. In AMI, a sudden blockage of blood flow causes myocardial ischemia and cell death. Reperfusion after ischemia has paradoxical effects and may exacerbate the myocardial injury, a process known as ischemic reperfusion injury. In this work we evaluated the lipidome of isolated rat hearts, maintained in controlled perfusion (CT), undergoing global ischemia (ISC) or ischemia followed by reperfusion (IR). 153 polar lipid levels were significantly different between conditions. 48 features had q < 0.001 and included 8 phosphatidylcholines and 4 lysophospholipids, which were lower in ISC compared to CT, and even lower in the IR group, suggesting that IR induces more profound changes than ISC. We observed that the levels of 16 alkyl acyl phospholipids were significantly altered during ISC and IR. Overall, these data indicate that myocardial lipid remodelling and possibly damage occurs to a greater extent during reperfusion. The adaptation of cardiac lipidome during ISC and IR described is consistent with the presence of oxidative damage and may reflect the impact of AMI on the lipidome at the cellular level and provide new insights into the role of lipids in the pathophysiology of acute myocardial ischemia/reperfusion injury.     

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

Introduction  

Vasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.     

A human cancer-predisposing polymorphism in Cdc25A is embryonic lethal in the mouse and promotes ASK-1 mediated apoptosis

Background

Failure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-β-TrCP (SCF^β-TrCP) complex. This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur.

Results

We now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both in vitro and in cultured cells. It fails to interact with the apoptosis signal-regulating kinase 1 (ASK1), however, and therefore does not suppress ASK1-mediated apoptosis.

Conclusions

These data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis.

     

Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension

Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.     

Impaired overload-induced hypertrophy is associated with diminished mTOR signaling in insulin-resistant skeletal muscle of the obese Zucker rat

Recent data have suggested that insulin resistance may be associated with a diminished ability of skeletal muscle to undergo hypertrophy (Paturi S, Gutta AK, Kakarla SK, Katta A, Arnold EC, Wu M, Rice KM, Blough ER. J Appl Physiol 108: 7-13, 2010). Here we examine the effects of insulin resistance using the obese Zucker (OZ) rat with increased muscle loading on the regulation of the mammalian target of rapamycin (mTOR) and its downstream signaling intermediates 70-kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6), eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Compared with that observed in lean Zucker (LZ) rats, the degree of soleus muscle hypertrophy as assessed by changes in muscle wet weight (LZ: 35% vs. OZ: 16%) was significantly less in the OZ rats after 3 wk of muscle overload (P < 0.05). This diminished growth in the OZ rats was accompanied by significant impairments in the ability of the soleus to undergo phosphorylation of mTOR (Ser(2448)), p70S6k (Thr(389)), rpS6 (Ser(235/236)), and protein kinase B (Akt) (Ser(473) and Thr(308)) (P < 0.05). Taken together, these data suggest that impaired overload-induced hypertrophy in insulin-resistant skeletal muscle may be related to decreases in the ability of the muscle to undergo mTOR-related signaling.     

Reduced plasma APOA1 level is associated with Gastric Tumor Growth in MKN45 mouse xenograft model

There is no suitable diagnostic and prognostic biomarker for gastric cancer. The biggest hurdles in biomarker discovery are (i) the low abundance of cancer cell-specific proteins that limits their detection and (ii) complex inter-patient variations that complicate the discovery process. To circumvent these issues, we conducted proteomics on the plasma of gastric cancer mouse xenograft and attempted to identify proteins released by cancer cells. MKN45 gastric cancer cells were subcutaneously implanted into immune-incompetent nude mice. Plasma samples collected from mice with different tumor sizes (low, mid and high tumor loads) were subjected to iTRAQ and mass spectrometric analyses. Detection of human APOA1 in mouse plasma was verified and its expression level was shown to be lower in mice with large tumors compared to those with small tumors. Studies on a panel of about 14 gastric cancer cell lines supported the notion that APOA1 in mouse plasma was of human gastric cancer cell origin. While the clinical utility of APOA1 remains to be ascertained with a larger scale study, the current work supported the feasibility of using mouse xenograft model for gastric cancer biomarker discovery.     

Hypercholesterolemia is associated with hyperactive cardiac mTORC1 and mTORC2 signaling

Nutritional excess and hyperlipidemia increase the heart’s susceptibility to ischemic injury. Mammalian target of rapamycin (mTOR) controls the cellular response to nutritional status and may play a role in ischemic injury. To explore the effect of hypercholesterolemia on cardiac mTOR signaling, we assessed mTOR signaling in hypercholesterolemic swine (HC) that are also susceptible to increased cardiac ischemia-reperfusion injury. Yucatan pigs were fed a high-fat/high-cholesterol diet for 4 weeks to induce hypercholesterolemia, and mTOR signaling was measured by immunoblotting and immunofluorescence in the non-ischemic left ventricular area. Total myocardial mTOR and raptor levels were markedly increased in the HC group compared to the normocholesterolemic group, and directly correlated with serum cholesterol levels. mTOR exhibited intense perinuclear staining in myocytes only in the HC group. Hypercholesterolemia was associated with hyperactive signaling upstream and downstream of both mTOR complexes, including myocardial Akt, S6K1, 4EBP1, S6, and PKC-alpha, increased levels of cardiac hypertrophy markers, and a trend toward lower levels of myocardial autophagy. Hypercholesterolemia can now be added to the growing list of conditions associated with aberrant mTOR signaling. Hypercholesterolemia produces a unique profile of alterations in cardiac mTOR signaling, which is a potential target in cardiac diseases associated with hypercholesterolemia and nutritional excess.     

Overgrowth of a mouse model of the Simpson-Golabi-Behmel syndrome is independent of IGF signaling

The type 1 Simpson-Golabi-Behmel overgrowth syndrome (SGBS1) is caused by loss-of-function mutations of the X-linked GPC3 gene encoding glypican-3, a cell-surface heparan sulfate proteoglycan that apparently plays a negative role in growth control by an unknown mechanism. Mice carrying a Gpc3 gene knockout exhibited several phenotypic features that resemble clinical hallmarks of SGBS1, including somatic overgrowth, renal dysplasia, accessory spleens, polydactyly, and placentomegaly. In Gpc3/DeltaH19 double mutants (lacking GPC3 and also carrying a deletion around the H19 gene region that causes bialellic expression of the closely linked Igf2 gene by imprint relaxation), the Gpc3-null phenotype was exacerbated, while additional SGBS1 features (omphalocele and skeletal defects) were manifested. However, results from a detailed comparative analysis of growth patterns in double mutants lacking GPC3 and also IGF2, IGF1, or the type 1 IGF receptor (IGF1R) provided conclusive genetic evidence inconsistent with the hypothesis that GPC3 acts as a growth suppressor by sequestering or downregulating an IGF ligand. Nevertheless, our data are compatible with a model positing that there is downstream convergence of the independent signaling pathways in which either IGFs or (indirectly) GPC3 participate.     

Endotoxemia is associated with altered innate and adaptive immune responses in untreated HIV-1 infected individuals

Background

Microbial translocation may contribute to the immunopathogenesis in HIV infection. We investigated if microbial translocation and inflammation were associated with innate and adaptive immune responses in adults with HIV.

Methodology/Principal Findings

This was an observational cohort study. Sera from HIV-infected and HIV-uninfected individuals were analyzed for microbial translocation (soluble CD14, lipopolysaccharides [LPS], endotoxin core antibody, and anti-α-galactosyl antibodies) and inflammatory markers (high sensitivity C-reactive protein, IL-6, IL-1 receptor antagonist, soluble tumor necrosis factor receptor II, and IL-10) with enzyme-linked immunosorbent assays. Peripheral blood mononuclear cells (PBMC) from HIV-infected persons and healthy controls (primed with single-stranded HIV-1-derived RNA) were stimulated with LPS, and cytokine production was measured. Finally, HIV-infected patients were immunized with Prevnar 7vPnC±CpG 7909 followed by Pneumo Novum PPV-23. Effects of microbial translocation and inflammation on immunization were analyzed in a predictive regression model. We included 96 HIV-infected individuals, 76 on highly active antiretroviral therapy (HAART), 20 HAART-naive, and 50 healthy controls. Microbial translocation and inflammatory markers were higher among HIV-infected persons than controls. Cytokine levels following LPS stimulation were increased in PBMCs from HAART-naive compared to HAART-treated HIV-infected persons. Further, RNA-priming of PBMCs from controls acted synergistically with LPS to augment cytokine responses. Finally, high serum LPS levels predicted poor vaccine responses among HAART-naive, but not among HAART-treated HIV-infected individuals.

Conclusions/Significance

LPS acts synergistically with HIV RNA to stimulate innate immune responses in vitro and increasing serum LPS levels seem to predict poor antibody responses after vaccination among HAART-naive HIV-infected persons. Thus, our results suggest that microbial translocation may be associated with innate and adaptive immune dysfunction in untreated HIV infection.     

Dopamine release is impaired in a mouse model of DYT1 dystonia

Early onset torsion dystonia, the most common form of hereditary primary dystonia, is caused by a mutation in the TOR1A gene, which codes for the protein torsinA. This form of dystonia is referred to as DYT1. We have used a transgenic mouse model of DYT1 dystonia [human mutant-type (hMT)1 mice] to examine the effect of the mutant human torsinA protein on striatal dopaminergic function. Analysis of striatal tissue dopamine (DA) and metabolites using HPLC revealed no difference between hMT1 mice and their non-transgenic littermates. Pre-synaptic DA transporters were studied using in vitro autoradiography with [(3)H]mazindol, a ligand for the membrane DA transporter, and [(3)H]dihydrotetrabenazine, a ligand for the vesicular monoamine transporter. No difference in the density of striatal DA transporter or vesicular monoamine transporter binding sites was observed. Post-synaptic receptors were studied using [(3)H]SCH-23390, a ligand for D(1) class receptors, [(3)H]YM-09151-2 and a ligand for D(2) class receptors. There were again no differences in the density of striatal binding sites for these ligands. Using in vivo microdialysis in awake animals, we studied basal as well as amphetamine-stimulated striatal extracellular DA levels. Basal extracellular DA levels were similar, but the response to amphetamine was markedly attenuated in the hMT1 mice compared with their non-transgenic littermates (253 +/- 71% vs. 561 +/- 132%, p < 0.05, two-way anova). These observations suggest that the mutation in the torsinA protein responsible for DYT1 dystonia may interfere with transport or release of DA, but does not alter pre-synaptic transporters or post-synaptic DA receptors. The defect in DA release as observed may contribute to the abnormalities in motor learning as previously documented in this transgenic mouse model, and may contribute to the clinical symptoms of the human disorder.     

Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.     

Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors

Introduction

Ankylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression.

Methods

PGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling.

Results

Inflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated.

Conclusions

This study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process.     

Abnormal notochord branching is associated with foregut malformations in the adriamycin treated mouse model

Oesophageal atresia (OA) and tracheooesophageal fistula (TOF) are relatively common human congenital malformations of the foregut where the oesophagus does not connect with the stomach and there is an abnormal connection between the stomach and the respiratory tract. They require immediate corrective surgery and have an impact on the future health of the individual. These abnormalities are mimicked by exposure of rat and mouse embryos in utero to the drug adriamycin. The causes of OA/TOF during human development are not known, however a number of mouse mutants where different signalling pathways are directly affected, show similar abnormalities, implicating multiple and complex signalling mechanisms. The similarities in developmental outcome seen in human infants and in the adriamycin treated mouse model underline the potential of this model to unravel the early embryological events and further our understanding of the processes disturbed, leading to such abnormalities. Here we report a systematic study of the foregut and adjacent tissues in embryos treated with adriamycin at E7 and E8 and analysed between E9 and E12, comparing morphology in 3D in 149 specimens. We describe a spectrum of 8 defects, the most common of which is ventral displacement and branching of the notochord (in 94% of embryos at E10) and a close spatial correspondence between the site of notochord branching and defects of the foregut. In addition gene expression analysis shows altered dorso-ventral foregut patterning in the vicinity of notochord branches. This study shows a number of features of the adriamycin mouse model not previously reported, implicates the notochord as a primary site of disturbance in such abnormalities and underlines the importance of the model to further address the mechanistic basis of foregut congenital abnormalities.     

Hyperlipidemia is associated with altered levels of insulin-like growth factor-I

Previous studies revealed altered levels of the circulating insulin-like growth factor-I (IGF-I) and of its binding protein-3 (IGFBP-3) in subjects with coronary atherosclerosis, metabolic syndrome and premature atherosclerosis. Hyperlipidemia is a powerful risk factor of atherosclerosis. We expected IGF-I and IGFBP-3 alterations in subjects with moderate/severe hyperlipidemia but without any clinical manifestation of atherosclerosis. Total IGF-I and IGFBP-3 were assessed in 56 patients with mixed hyperlipidemia (MHL; cholesterol >6.0 mmol/l, triglycerides >2.0 mmol/l), in 33 patients with isolated hypercholesterolemia (IHC; cholesterol >6.0 mmol/l, triglycerides <2.0 mmol/l), and in 29 healthy controls (cholesterol<6.0 mmol/l, triglycerides<2.0 mmol/l). The molar ratio of IGF-I/IGFBP-3 was used as a measure of free IGF-I. IHC subjects differed from controls by lower total IGF-I (164+/-60 vs. 209+/-73 ng/ml, p=0.01) and IGF-I /IGFBP-3 ratio (0.14+/-0.05 vs. 0.17+/-0.04, p=0.04). Compared to controls, MHL subjects had lower total IGF-I (153+/-54 ng/ml, p=0.0002) and IGFBP-3 (2.8+/-0.6 mg/ml, p<0.0001), but higher IGF-I/IGFBP-3 ratio (0.25+/-0.06, p<0.0001). Differences remained significant after the adjustment for clinical and biochemical covariates, except for triglycerides. Patients with both IHC and MHL have lower total IGF-I compared to controls. The mechanism is presumably different in IHC and MHL. Because of prominent reduction of IGFBP-3 in patients with MHL, they have reduced total IGF-I despite the actual elevation IGF-I/IGFBP-3 ratio as a surrogate of free IGF-I.     

Gpx1 is a stationary phase-specific thioredoxin peroxidase in fission yeast

The genome sequence of Schizosaccharomyces pombe reveals only one gene for a putative glutathione peroxidase (gpx1⁺). The Gpx1 protein has a peroxidase activity but preferred thioredoxin to glutathione as an electron donor when examined in vitro and in vivo, and therefore is a thioredoxin peroxidase. Besides H₂O₂, it can reduce alkyl and phospholipid hydroperoxides. Expression of the gpx1 gene was elevated at the stationary phase, and we found that it supported long-term survival of S. pombe. The mutant also exhibited some defect in the activity of aconitase, an oxidation-labile Fe-S enzyme in mitochondria. Activity of sulfite reductase, a labile Fe-S enzyme in the cytosol, was also dramatically lowered in the mutant in the stationary phase. The Gpx1 protein, without any obvious targeting sequence, was localized in mitochondria as well as in the cytosol. Therefore, Gpx1 must serve to ensure optimal mitochondrial function and cytosolic environment, especially in the stationary phase.