Murugesan Velayutham

A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal

The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base—a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signals interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6′ (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.

Cardiac Myocyte–Specific Expression of Inducible Nitric Oxide Synthase Protects Against Ischemia/Reperfusion Injury by Preventing Mitochondrial Permeability Transition

Background— Inducible nitric oxide synthase (iNOS) is an obligatory mediator of the late phase of ischemic preconditioning, but the mechanisms of its cardioprotective actions are unknown. In addition, it remains unclear whether sustained elevation of iNOS in myocytes provides chronic protection against ischemia/reperfusion injury. Methods and Results— Constitutive overexpression of iNOS in transgenic mice (α-myosin heavy chain promoter) did not induce contractile dysfunction and did not affect mitochondrial respiration or biogenesis, but it profoundly decreased infarct size in mice subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion. In comparison with wild-type hearts, isolated iNOS-transgenic hearts subjected to ischemia for 30 minutes followed by 40 minutes of reperfusion displayed better contractile recovery, smaller infarct size, and less mitochondrial entrapment of 2-deoxy-[3H]-glucose. Reperfusion-induced loss of NAD+ and mitochondrial release of cytochrome c were attenuated in iNOS-transgenic hearts, indicating reduced mitochondrial permeability transition. The NO donor NOC-22 prevented permeability transition in isolated mitochondria, and mitochondrial permeability transition–induced NAD+ loss was decreased in wild-type but not iNOS-null mice treated with the NO donor diethylene triamine/NO 24 hours before ischemia and reperfusion ex vivo. iNOS-mediated cardioprotection was not abolished by atractyloside. Reperfusion-induced production of oxygen-derived free radicals (measured by electron paramagnetic resonance spectroscopy) was attenuated in iNOS-transgenic hearts and was increased in wild-type hearts treated with the mitochondrial permeability transition inhibitor cyclosporin A. Conclusions— Cardiomyocyte-restricted expression of iNOS provides sustained cardioprotection. This cardioprotection is associated with a decrease in reperfusion-induced oxygen radicals and inhibition of mitochondrial swelling and permeability transition.

In vivo atherosclerotic plaque characterization using magnetic susceptibility distinguishes symptom-producing plaques.

OBJECTIVES We investigated the role of iron deposition in atherosclerotic plaque instability using a novel approach of in vivo plaque characterization by a noninvasive, noncontrast magnetic resonance-based T2* measurement. This approach was validated using ex vivo plaque analyses to establish that T2* accurately reflects intraplaque iron composition. BACKGROUND Iron catalyzes free radical production, a key step for lipid peroxidation and atherosclerosis development. The parameter T2* measures tissue magnetic susceptibility, which historically has been used to quantify hepatic and myocardial iron. The T2* measurement has not been used for in vivo plaque characterization in patients with atherosclerosis. METHODS Thirty-nine patients referred for carotid endarterectomy were prospectively enrolled to undergo preoperative carotid magnetic resonance imaging (MRI) and postoperative analysis of the explanted plaque. Clinical history of any symptoms attributable to each carotid lesion was recorded. We could not complete MRI in 4 subjects because of their claustrophobia, and 3 patients scanned before the institution of a neck stabilizer had motion artifact, precluding quantification. RESULTS Symptomatic patients had significantly lower plaque T2* values (20.0 +/- 1.8 ms) compared with asymptomatic patients (34.4 +/- 2.7 ms, p < 0.001). Analytical methods demonstrated similar total iron (138.6 +/- 36.5 microg/g vs. 165.8 +/- 48.3 microg/g, p = NS) but less low molecular weight Fe(III) (7.3 +/- 3.8 microg/g vs. 17.7 +/- 4.0 microg/g, p < 0.05) in the explanted plaques of symptomatic versus asymptomatic patients, respectively, which is consistent with a shift in iron from Fe(III) to greater amounts of T2*-shortening forms of iron. Mass spectroscopy also showed significantly lower calcium (37.5 +/- 10.8 mg/g vs. 123.6 +/- 19.3 mg/g, p < 0.01) and greater copper (3.2 +/- 0.5 microg/g vs. 1.7 +/- 0.1 microg/g, p < 0.01) in plaques from symptomatic patients. CONCLUSIONS In vivo measurement of intraplaque T2* using MRI is feasible and distinguishes symptom-producing from non-symptom-producing plaques in patients with carotid artery atherosclerosis. Symptom-producing plaques demonstrated characteristic changes in iron forms by ex vivo analysis, supporting the dynamic presence of iron in the microenvironment of atherosclerotic plaque.

Aldehyde Oxidase Functions as a Superoxide Generating NADH Oxidase: An Important Redox Regulated Pathway of Cellular Oxygen Radical Formation

The enzyme aldehyde oxidase (AO) is a member of the molybdenum hydroxylase family that includes xanthine oxidoreductase (XOR); however, its physiological substrates and functions remain unclear. Moreover, little is known about its role in cellular redox stress. Utilizing electron paramagnetic resonance spin trapping, we measured the role of AO in the generation of reactive oxygen species (ROS) through the oxidation of NADH and the effects of inhibitors of AO on NADH-mediated superoxide (O(2)(•−)) generation. NADH was found to be a good substrate for AO with apparent K(m) and V(max) values of 29 μM and 12 nmol min(-1) mg(-1), respectively. From O(2)(•−) generation measurements by cytochrome c reduction the apparent K(m) and V(max) values of NADH for AO were 11 μM and 15 nmol min(-1) mg(-1), respectively. With NADH oxidation by AO, ≥65% of the total electron flux led to O(2)(•−) generation. Diphenyleneiodonium completely inhibited AO-mediated O(2)(•−) production, confirming that this occurs at the FAD site. Inhibitors of this NADH-derived O(2)(•−) generation were studied with amidone the most potent exerting complete inhibition at 100 μM concentration, while 150 μM menadione, raloxifene, or β-estradiol led to 81%, 46%, or 26% inhibition, respectively. From the kinetic data, and the levels of AO and NADH, O(2)(•−) production was estimated to be ~89 and ~4 nM/s in liver and heart, respectively, much higher than that estimated for XOR under similar conditions. Owing to the ubiquitous distribution of NADH, aldehydes, and other endogenous AO substrates, AO is predicted to have an important role in cellular redox stress and related disease pathogenesis.

Tetrahydrobiopterin depletion and NOS2 uncoupling contribute to heart failure-induced alterations in atrial electrophysiology

AIMS Heart failure is a common antecedent to atrial fibrillation; both heart failure and atrial fibrillation are associated with increased myocardial oxidative stress. Chronic canine heart failure reduces atrial action potential duration and atrial refractoriness. We hypothesized that inducible nitric oxide synthase 2 (NOS2) contributes to atrial oxidative stress and electrophysiologic alterations. METHODS AND RESULTS A 16-week canine tachypacing model of heart failure was used (n= 21). At 10 weeks, dogs were randomized to either placebo (n = 12) or active treatment (n = 9) with NOS cofactor, tetrahydrobiopterin (BH(4), 50 mg), and NOS substrate (L-arginine, 3 g) twice daily for 6 weeks. A group of matched controls (n = 7) was used for comparison. Heart failure increased atrial NOS2 and reduced atrial BH(4), while L-arginine was unchanged. Treatment reduced inducible atrial fibrillation and normalized the heart failure-induced shortening of the left atrial myocyte action potential duration. Treatment increased atrial [BH(4)] while [L-arginine] was unchanged. Treatment did not improve left ventricular function or dimensions. Heart failure-induced reductions in atrial [BH(4)] resulted in NOS uncoupling, as measured by NO and superoxide anion (O(2)(·-)) production, while BH(4) and L-arginine treatment normalized NO and O(2)(·-). Heart failure resulted in left atrial oxidative stress, which was attenuated by BH(4) and L-arginine treatment. CONCLUSION Chronic non-ischaemic heart failure results in atrial oxidative stress and electrophysiologic abnormalities by depletion of BH(4) and uncoupling of NOS2. Modulation of NOS2 activity by repletion of BH(4) may be a safe and effective approach to reduce the frequency of atrial arrhythmias during heart failure.

Early ischaemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation

AIMS The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC. METHODS AND RESULTS Rat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOS(Ser1176), Akt(Ser473), and PKA(Thr197) phosphorylation was increased following IPC. I/R decreased eNOS(Ser1176) phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOS(Ser1176) phosphorylation and quantitative Western blots showed ∼24% modification of eNOS(Ser1176) following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased Akt(Ser473) or PKA(Thr197) phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOS(Ser1176) phosphorylation as well as cardioprotection. CONCLUSION Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role.

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.

EPR oxygen mapping (EPROM) of engineered cartilage grown in a hollow-fiber bioreactor

A novel electron paramagnetic resonance (EPR)‐based oxygen mapping procedure (EPROM) is applied to cartilage grown in a single‐, hollow‐fiber bioreactor (HFBR) system. Chondrocytes harvested from the sterna of 17‐day‐old chick embryos were inoculated into an HFBR and produced hyaline cartilage over a period of 4 weeks. Tissue oxygen maps were generated according to the EPROM technique (Velan et al., Magn Reson Med 2000;43:804–809) by making use of the line‐broadening effects of oxygen on the signal generated from nitroxide spin probes. In addition, the effect on oxygen consumption of the addition of cyanide to the tissue was investigated. Cyanide is a potent inhibitor of oxidative phosphorylation, and accordingly, given the constant provision of oxygen to the tissue, it would be expected to increase oxygen levels within the HFBR. The EPROM measurements showed a significant increase in oxygen concentration in the cartilage after the addition of cyanide. In contrast to other methods for studying oxygen in cartilage, EPROM can provide direct, noninvasive visualization of local concentrations in three dimensions. Magn Reson Med 46:819–826, 2001.

Hydrogen peroxide is a second messenger in phase 2 enzyme induction by cancer chemopreventive dithiolethiones

The ability of three dithiolethione cancer chemopreventives, oltipraz 1, anetholedithione (ADT) 2, 1,2-dithiole-3-thione (D3T) 3, and the major metabolite, 4, of 1, to induce the cytoprotective enzyme NQO1 in Hepa 1c1c7 cells and the inhibition of this induction by catalase are demonstrated. The ability of 1, 3, and 4 to form O(2)(*) has been reported, and it is here demonstrated that 2 decomposes in the presence of GSH to form, upon addition of the nitrone spin trap DMPO, the DMPO-OH adduct that is detectable by EPR. Decomposition of 2 in the presence of GSH elicits, upon the addition of hydroethidine and excitation at 510 nm, fluorescence at 580 nm that is diminished by the addition of superoxide dismutase. The compound 4, is a product of the reduction of 1, and it is demonstrated that 2 and 3 decompose in the presence of reductants such as thiolates and NaBH(4), followed by addition of CH(3)I, to form the dimethylated products of reductive cleavage of the S(1)-S(2) bond. The same products are isolated subsequent to lysis in buffer containing CH(3)I of Hepa 1c1c7 cells treated with 2 or 3. Reductive cleavage of 2 and 3 in aqueous ethanol by NaBH(4) in an argon atmosphere, followed by acidic destruction of remaining borohydride and neutralization and introduction of O(2) results in the reformation of 2 and 3 to the extent of 80 and 33%, respectively. The data in toto are consistent with a model in which dithiolethiones, generally, undergo reductive cleavage in Hepa 1c1c7 cells, thereby resulting in the generation of O(2)(*) that dismutates to H(2)O(2), that subsequently, by direct or indirect means, effects the nuclear translocation of transcription factor Nrf2, that upregulates phase 2 enzyme expression.

Biphasic effect of SIN-1 is reliant upon cardiomyocyte contractile state.

Many studies have demonstrated a biphasic effect of peroxynitrite in the myocardium, but few studies have investigated this biphasic effect on beta-adrenergic responsiveness and its dependence on contractile state. We have previously shown that high 3-morpholinosydnonimine (SIN-1) (source of peroxynitrite, 200 micromol/L) produced significant anti-adrenergic effects during maximal beta-adrenergic stimulation in cardiomyocytes. In the current study, we hypothesize that the negative effects of high SIN-1 will be greatest during high contractile states, whereas the positive effects of low SIN-1 (10 micromol/L) will predominate during low contractility. Isolated murine cardiomyocytes were field stimulated at 1 Hz, and [Ca(2+)](i) transients and shortening were recorded. After submaximal isoproterenol (ISO) (beta-adrenergic agonist, 0.01 micromol/L) stimulation, 200 micromol/L SIN-1 induced two distinct phenomena. Cardiomyocytes undergoing a large response to ISO showed a significant reduction in contractility, whereas cardiomyocytes exhibiting a modest response to ISO showed a further increase in contractility. Additionally, 10 micromol/L SIN-1 always increased contractility during low ISO stimulation, but had no effect during maximal ISO (1 micromol/L) stimulation. SIN-1 at 10 micromol/L also increased basal contractility. Interestingly, SIN-1 produced a contractile effect under only one condition in phospholamban-knockout cardiomyocytes, providing a potential mechanism for the biphasic effect of peroxynitrite. These results provide clear evidence for a biphasic effect of peroxynitrite, with high peroxynitrite modulating high levels of beta-adrenergic responsiveness and low peroxynitrite regulating basal function and low levels of beta-adrenergic stimulation.