Irreversible Protein Oxidation Research Articles

The location of protein oxidation in dystrophic skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

A10274 Interplay between Nox-regulated oxidative stress and ER stress response in experimental hypertension

Objectives: Nox-derived ROS and ER stress are implicated in vascular dysfunction in hypertension. Evidence suggests a cross-talk between these processes; however the mechanisms involved are still unknown. We investigated how oxidative stress impacts the oxidative proteome and the ER stress response in VSMC from SHR rats. Methods: Nox compartmentalization (cell fractionation), O2- (lucigenin), H2O2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase (PTP), peroxiredoxin (Prx) oxidation) and ER stress (PERK, IRE1α, phosphorylation/oxidation) were assessed in SHR VSMC. VSMC proliferation was measured by FACS and vascular reactivity by myography. Results: High levels of O2- (67%) and H2O2 (204%) were observed in SHR, p < 0.05 vs WKY. Reversible and irreversible protein oxidation were also increased in SHR (p < 0.05 vs WKY). ER stress was upregulated in SHR, as phosphorylation of PERK and IRE1α were increased (p < 0.05 vs WKY). Noxs were upregulated in a compartment-specific manner in SHR: Nox1 in plasma membrane (1.6 fold) and Nox4 in ER (1.3 fold, p < 0.05 vs WKY). Inhibition of Nox1 (NoxA1ds) and Nox1/4 (GKT137831) normalized vascular ROS generation and protein oxidation in SHR. NoxA1ds reduced PERK phosphorylation, while GKT137831 reduced IRE1α sulfenylation. Inhibition of ER stress (4-PBA) reduced ROS levels and expression of Nox4 in SHR. Increased VSMC proliferation in SHR (51%, p < 0.05 vs WKY) was normalized by GKT137831 and 4-PBA. Vascular hypercontractility, assessed in mesenteric arteries, was attenuated by 4-PBA. Conclusion: Our findings demonstrate that in hypertension oxidative stress is associated with ER stress through processes involving Nox decompartmentalization, where Nox1 mediated activation of PERK and Nox4 induced oxidation of IRE1α.

Vascular Nox (NADPH Oxidase) Compartmentalization, Protein Hyperoxidation, and Endoplasmic Reticulum Stress Response in Hypertension.

Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O2- (lucigenin), H2O2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1α [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1α sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1-XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1-XBP1 pathway of the ER stress response is regulated by Nox4/reactive oxygen species and plays a role in the hyperproliferative VSMC phenotype in SHR. Our study highlights the importance of Nox subcellular compartmentalization and interplay between cytoplasmic reactive oxygen species and ER stress response, which contribute to the VSMC oxidative proteome and vascular dysfunction in hypertension.

A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma

Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (−32Da) - and decarboxylated glutamic acid and aspartic acid (−30Da). Biological significanceProteomic tools provide a promising way to decode disease mechanisms at the protein level and help to understand how carbonylation affects protein structure and function. The challenge for future research is to identify the type and nature of oxidised residues to gain a deeper understanding of the mechanism(s) governing carbonylation in cells and organisms and assess their role in disease.

Assessment of Protein Carbonylation and Protein Tyrosine Phosphatase (PTP) Oxidation in Vascular Smooth Muscle Cells (VSMCs) Using Immunoblotting Approaches.

Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irreversible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome of vascular smooth muscle cells (VSMC).Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible protein carbonylation in the sample by immunoblotting.Oxidation of PTPs can be evaluated using a monoclonal antibody against the "hyperoxidized" (SO3H) catalytic site of these enzymes. The described methodology offers the ability to discriminate between irreversible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S-) are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both untreated and PV-treated samples are analyzed by SDS-PAGE and "hyperoxidized" PTPs are detected by immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference between the signals in untreated and the PV-treated samples.The above immunoassays provide general approaches to detect and quantify global levels of irreversible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context. Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms underlying chronic diseases associated with oxidative stress. This is particularly important in systems influenced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox signaling.

Protein glutathionylation protects wheat (Triticum aestivum Var. Sonalika) against Fusarium induced oxidative stress

Fusarium induced oxidative stress could be recovered by reversible protein oxidative modification through the process of glutathionylation in co-stressed (low-dose (50 μM) Cd2+ pre-treatment followed by Fusarium inoculation) wheat seedlings. Co-stressed seedlings showed low disease severity index as compared to Fusarium infected seedlings. A reduced level of hydrogen peroxide (H2O2) and carbonyl contents due to irreversible protein oxidation were observed in co-stressed seedlings as compared to Fusarium infected seedlings. Further, a comparative biochemical assay showed an enhanced glutathione content in co-stressed tissues as compared to Fusarium infected tissues. In an investigation, reduced glutathione pre-coated agarose gel beads were used to pull down proteins having affinity with GSH. Fructose-1, 6-bisphosphate aldolase and 3-Phosphoglycerate kinase were observed to be co-existed in co-stressed seedlings when analysed by LC-MS/MS after being processed through protein-pull assay. Co-stressed tissues showed an enhanced free protein thiol content as compared to Fusarium infected tissues. The ratio of free thiol to thiol disulfides was also observed to be increased in co-stressed tissues as compared to Fusarium infected tissues. In contrast, the quantitative assay by Ellman's reagent and qualitative analysis by diagonal gel electrophoresis showed enhanced protein thiol disulfides in Fusarium infected tissues as compared to co-stressed tissues. Further, glutaredoxin, responsible for the reverse reduction of proteins was observed to be enhanced in co-stressed tissues as compared to Fusarium infected tissues. Thus, a low dose Cd2+ triggered glutathionylation is suggestive of offering tolerance against Fusarium induced oxidative stress and protects target proteins from irreversible modification and permanent damage in wheat.

41 Vascular protein oxidation and redox proteomics in human hypertension

Despite recent advances in the field of vascular redox signalling in hypertension, it still remains unclear exactly how ROS cause vascular injury. We hypothesise that regulation of redox-sensitive protein tyrosine phosphatases (PTP) through oxidative modification, is impaired in hypertension. VSMC normotensive (NT) and hypertensive (HT) individuals were stimulated with AngII (10-7 M) and ET-1 (10-7 M). Irreversible oxidation of proteins and PTPs was assessed by oxyblot. Differential gel electrophoresis (DiGE) and CyDye thiol labelling were employed for screening of reversibly oxidised proteome. Irreversible protein oxidation was not affected by AngII or ET-1 in VSMCs from NT and HT subjects. Proteomic data, filtered for FC >2, detected 2051 spots with 1899 (92.5%) being equally oxidised between NT and HT. In addition, oxidation of 57 (2.9%) spots was increased, while 95 (4.6%) were decreased in HT. Candidate proteins exhibiting consistent changes across three experimental replicates included β-actin (FC = –2.86), annexin A1 (–2.23), galectin-1 (–1.67), FK506 binding protein (–2.35) and polymerase I and transcript release factor (PTRF, –1.92). Stimulation with AngII altered the redox status in 2–3% of proteins, both in HT and NT. However, vimentin was the only target changing consistently across the replicates (FC = 2.48). Our findings indicate that pro-hypertensive agents may not impact significantly on irreversible protein and PTP oxidation in health and disease, but may have effects on reversible oxidation. Our proteomic data, in agreement with our previous rat studies, support decreased reversible thiol oxidation in hypertension.

S-Glutathionylation and Redox Protein Signaling in Drug Addiction.

Drug addiction is a chronic relapsing disorder that comes at a high cost to individuals and society. Therefore understanding the mechanisms by which drugs exert their effects is of prime importance. Drugs of abuse increase the production of reactive oxygen and nitrogen species resulting in oxidative stress. This change in redox homeostasis increases the conjugation of glutathione to protein cysteine residues; a process called S-glutathionylation. Although traditionally regarded as a protective mechanism against irreversible protein oxidation, accumulated evidence suggests a more nuanced role for S-glutathionylation, namely as a mediator in redox-sensitive protein signaling. The reversible modification of protein thiols leading to alteration in function under different physiologic/pathologic conditions provides a mechanism whereby change in redox status can be translated into a functional response. As such, S-glutathionylation represents an understudied means of post-translational protein modification that may be important in the mechanisms underlying drug addiction. This review will discuss the evidence for S-glutathionylation as a redox-sensing mechanism and how this may be involved in the response to drug-induced oxidative stress. The function of S-glutathionylated proteins involved in neurotransmission, dendritic spine structure, and drug-induced behavioral outputs will be reviewed with specific reference to alcohol, cocaine, and heroin.

Abstract P096: Vascular Protein Oxidation and Redox Proteomics in Human Hypertension

Oxidative stress has been implicated in the pathophysiology of hypertension (HTN) through redox-sensitive processes that cause vascular damage. Despite recent advances in the field of vascular redox signalling in HTN, it still remains unclear exactly how ROS cause vascular injury. We hypothesise that regulation of redox-sensitive protein tyrosine phosphatases (PTP) through post-translational oxidative modification, is impaired in HTN where ROS levels are increased. Vascular smooth muscle cells (VSMC) from small arteries of normotensive (NT) and hypertensive (HT) individuals were stimulated with AngII (10-7 M) and ET-1 (10-7 M). Irreversible oxidation of proteins and PTPs was assessed by oxyblot and immunoblotting, respectively. Differential gel electrophoresis (DiGE) and CyDye thiol labelling were employed for screening of reversibly oxidised proteome. Irreversible protein oxidation was not affected by AngII or ET-1 in VSMCs from NT and HT subjects. PTP hyperoxidation tended to increase in VSMCs from NT upon stimulation with AngII (FC=2.12 at 60min) and ET-1 (FC=1.60 at 60min), whereas a similar trend was observed only after AngII treatment (FC=1.38 at 60min) in HTN (p&gt;0.05). Proteomic data, filtered for FC&gt;2, detected 2051 spots with 1899 (92.5%) being equally oxidised between NT and HT. In addition, oxidation of 57 (2.9%) spots was increased, while 95 (4.6%) were decreased in HT. Candidate proteins exhibiting consistent changes across three experimental replicates included β-actin (FC=-2.86), annexin A1 (-2.23), galectin-1 (-1.67), FK506 binding protein (-2.35) and polymerase I and transcript release factor (PTRF, -1.92). Stimulation with AngII altered the redox status in 2-3% of proteins, both in HT and NT. However, vimentin was the only target changing consistently across the replicates (FC=2.48). Our findings indicate that pro-hypertensive agents may not impact significantly on irreversible protein and PTP oxidation in health and disease, but may have effects on reversible oxidation. Our proteomic data, in agreement with our previous rat studies, support decreased reversible thiol oxidation in HTN. Moreover, these novel findings identify differentially oxidised proteins which may contribute to oxidative vascular injury in HTN.

Abstract 1825: Carbonylation and degradation of cardiac myosin binding protein C serves as an indicator of doxorubicin-induced cardiotoxicity

Abstract Dose-dependent oxidative stress by the anthracycline doxorubicin (Dox) and other chemotherapeutic agents causes irreversible cardiac damage, restricting their clinical effectiveness. We hypothesize that irreversible protein oxidation caused by oxidative stress could be correlated with physiological functional impairment. We focused our study on protein carbonylation as an indicator of severe oxidative damage. We identified a specific cardiac protein that showed selective carbonylation under Dox-induced cardiotoxic conditions in a spontaneously hypertensive rat (SHR) model and this protein was confirmed to be a 140 kDa cardiac myosin binding protein C (MyBPC) using mass spectrometry. We further analyzed and confirmed the carbonylation and degradation of this protein using HL-1 cardiomyocytes under Dox-induced oxidative stress, and a purified recombinant rat cardiac MyBPC under metal-catalyzed oxidative stress conditions. Using HL-1 cells we demonstrated that carbonylated MyBPC undergoes proteasome-mediated degradation under Dox-induced oxidative stress. Using immunoprecipitation, co-sedimentation, and actin-binding assays we confirmed the functional impairment of MyBPC as a consequence of carbonylation. Carbonylation of recombinant MyBPC showed significant impairment in its actin binding properties with a dissociation constant of 7.35±1.9 μM compared to 2.7±0.6 μM for native MyBPC. Overall, our findings indicate that MyBPC carbonylation serves as a critical determinant of cardiotoxicity. We are currently investigating the interaction of MyBPC with divalent metal ions and sites of carbonylation under oxidative stress conditions. Citation Format: Baikuntha P. Aryal, Jinsook Jeong, V. Ashutosh Rao. Carbonylation and degradation of cardiac myosin binding protein C serves as an indicator of doxorubicin-induced cardiotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1825. doi:10.1158/1538-7445.AM2015-1825

Unravelling the reactive oxygen and reactive nitrogen signalling networks in plants.

Unravelling the reactive oxygen and reactive nitrogen signalling networks in plants.

Protein carbonylation and metal-catalyzed protein oxidation in a cellular perspective

Proteins can become oxidatively modified in many different ways, either by direct oxidation of amino acid side chains and protein backbone or indirectly by conjugation with oxidation products of polyunsaturated fatty acids and carbohydrates. While reversible oxidative modifications are thought to be relevant in physiological processes, irreversible oxidative modifications are known to contribute to cellular damage and disease. The most well-studied irreversible protein oxidation is carbonylation. In this work we first examine how protein carbonylation occurs via metal-catalyzed oxidation (MCO) in vivo and in vitro with an emphasis on cellular metal ion homeostasis and metal binding. We then review proteomic methods currently used for identifying carbonylated proteins and their sites of modification. Finally, we discuss the identified carbonylated proteins and the pattern of carbonylation sites in relation to cellular metabolism using the mitochondrion as a case story.

The Effect of Glutathione on Skeletal Muscle Calcium Sensitivity and Myofilament Sulfhydryl Groups

Glutathione, a critical reducing agent present in relatively high levels(∼5mM) in skeletal muscle, can also attach to protein thiols via a disulfide bond. This process is referred to as glutathionylation, and is thought to be a protective mechanism to prevent irreversible protein oxidation. Prior studies have shown that when skinned fibers are exposed to reduced glutathione there is an increase in calcium sensitivity with no significant change in maximal force. These calcium sensitivity changes were largely reversible by the reducing agent DTT, indicating modification of protein thiols. We measured the force-pCa relationship of permeabilized rabbit psoas fibers treated with DTDP a thiol-specific oxidizing agent and glutathione (5mM). 2D gel electrophoresis using either IEF 4-6.5 or NEPHGE 3-10 in the first dimension was used to identify myofilament proteins whose sulfhydryl groups were modified with the oxidant-glutathione treatment. Additionally, phosphorylation of the regulatory myosin light chain was analyzed using 2d gels (IEF 4-6.5) and the phosphorylation specific stain Diamond Pro-Q. The pCa 50 of skinned psoas fibers was decreased upon exposure to DTT. Following DTT, the addition of DTDP and GSH sequentially, increased the calcium sensitivity. 2D gel electrophoresis, indicated myosin as a critical target for glutahionylation under these conditions. The RMLC was phosphorylated in the psoas fibers and the level remained constant during oxidant - glutathione treatment. In summary, our data suggest that glutathionylation of myofilament proteins can modulate calcium sensitivity, and may play an important role in maintaining muscle function during oxidative stress.

Reversible Intramolecular Hydrogen Transfer between Cysteine Thiyl Radicals and Glycine and Alanine in Model Peptides: Absolute Rate Constants Derived from Pulse Radiolysis and Laser Flash Photolysis

The intramolecular reaction of cysteine thiyl radicals with peptide and protein alphaC-H bonds represents a potential mechanism for irreversible protein oxidation. Here, we have measured absolute rate constants for these reversible hydrogen transfer reactions by means of pulse radiolysis and laser flash photolysis of model peptides. For N-Ac-CysGly6 and N-Ac-CysGly2AspGly3, Cys thiyl radicals abstract hydrogen atoms from Gly with k(f) = (1.0-1.1 x 10(5) s(-1), generating carbon-centered radicals, while the reverse reaction proceeds with k(r) = (8.0-8.9) x 10(5) s(-1). The forward reaction shows a normal kinetic isotope effect of k(H)/k(D) = 6.9, while the reverse reaction shows a significantly higher normal kinetic isotope effect of 17.6, suggesting a contribution of tunneling. For N-Ac-CysAla2AspAla3, cysteine thiyl radicals abstract hydrogen atoms from Ala with k(f) = (0.9-1.0) x 10(4) s(-1), while the reverse reaction proceeds with k(r) = 1.0 x 10(5) s(-1). The order of reactivity, Gly > Ala, is in accord with previous studies on intermolecular reactions of thiyl radicals with these amino acids. The fact that k(f) < k(r) suggests some secondary structure of the model peptides, which prevents the adoption of extended conformations, for which calculations of homolytic bond dissociation energies would have predicted k(f) > k(r). Despite k(f) < k(r), model calculations show that intramolecular hydrogen abstraction by Cys thiyl radicals can lead to significant oxidation of other amino acids in the presence of physiologic oxygen concentrations.

Abstract 924: Aging Induces Titin Oxidation And Increases Passive Cardiomyocyte Stiffness

One of the hallmarks of aging is an increase in ventricular passive stiffness leading to diastolic heart failure, however, the molecular basis for this is unclear. The giant elastic protein titin, working as an entropic spring, is the major determinant of passive cardiomyocyte stiffness. We hypothesized that accumulation of oxidized titin in the aging heart results in increased cardiomyocyte passive stiffness and thereby contributes to diastolic dysfunction. We further hypothesized that an age-assocated disturbance in the ubiquitin/proteasome pathway plays a role in the accumulation of oxidized titin. Methods: Cardiomyocytes from adult (4 month) and aging (34 month) mouse hearts were isolated and chemically skinned and the passive force-sarcomere length relationship in single cells were obtained. Post-translational modification of titin was assessed in whole heart homogenates by determining the content of 1) titin carbonyls, a marker of irreversible protein oxidation, and 2) titin ubiquitinylation, a marker for proteasomal degradation. Results: Cardiomyocyte passive tension was significantly higher in the aging group (at SL=2.45 μm, 1008.4 ± 45.5 vs. 871.6 ± 48.3 μg; p&lt;0.05). While the total amount of titin was unchanged between both groups, the carbonyl content of titin was increased 3-fold with aging (p&lt;0.05). High molecular weight ubiquitin-conjugated proteins were increased with aging, which is consistent with a decrease in proteasomal activity with age. Surprisingly, titin ubiquitinylation was decreased with aging suggesting an impairment upstream of the proteasome, in titin-specific ubiquitin ligase activity. Furthermore, the muscle specific ubiquitin ligase MURF-2, which has been shown to interact with titin and play a role in muscle protein turnover, showed a significant shift from a high to low molecular weight isoform with aging. Conclusion: Our data suggests that an age-associated impairment in titin turnover contributes to accumulation of oxidatively modified titins with consequent impairment of cardiomyocyte stiffness. We speculate that the oxidation of titin will change the elastic properties of titin, directly accounting for the change in passive tension and contributing to diastolic dysfunction associated with aging.

Inactivation of hepatic enzymes by inhalant nitrite—In vivo and in vitro studies

We examined the effects of acute isobutyl nitrite (ISBN) exposure on the activity of several hepatic enzymes. Two strains of adult male mice (Balb/c and C57BL/6) were exposed to 900 ppm ISBN or ambient air for 45 minutes. The enzyme activity of hepatic cytochrome P450 (CYP)-mediated deethylation, glutathione S-transferase (GST), and carboxylesterase (CBE) was monitored through the substrates 3-cyano-7-ethoxycoumarin (CEC), 1-chloro-2,4-dinitrobenzene, and p-nitrophenyl acetate, respectively. Acute ISBN exposure led to a significant reduction in hepatic CYP-mediated CEC deethylation, GST, and CBE activity in Balb/c mice (of 81.5%, 74.7%, and 25.2%, respectively, vs control mice, each at P < .05) when livers were harvested immediately after inhalant exposure. The corresponding decreases in C57BL/6 mice were smaller (with reductions of 21.8%, 18.8%, and 13.3%, respectively, each at P < .05). This enzyme activity, tested in C57BL/6 mice only, returned to control values after a 24-hour period of nonexposure. Follow-up mechanistic investigations using rat liver GST indicated that ISBN-mediated enzyme inactivation was not caused by its metabolites: inorganic nitrite ion (NO2-) or nitric oxide. This inactivation could be prevented, but not reversed, by added glutathione, suggesting irreversible protein oxidation. Using different NO donors as comparative agents, we found that GST inactivation by ISBN was not associated with protein S-nitrosylation or disulfide formation, but with tyrosine nitration. Inhalant nitrite exposure, therefore, led to a significant reduction in hepatic enzyme activity in mice, possibly through tyrosine nitration of hepatic proteins. This effect raises the possibility of drug-drug metabolic interactions from inhalant nitrite abuse. However, determining the applicability of these findings to humans will require further study.