Takotsubo syndrome is a coronary microvascular disease: experimental evidence.

BACKGROUND AND AIMS: Takotsubo syndrome (TTS) is a conundrum without consensus about the cause. In a murine model of coronary microvascular dysfunction (CMD), abnormalities in myocardial perfusion played a key role in the development of TTS. METHODS AND RESULTS: Vascular Kv1.5 channels connect coronary blood flow to myocardial metabolism and their deletion mimics the phenotype of CMD. To determine if TTS is related to CMD, wild-type (WT), Kv1.5-/-, and TgKv1.5-/- (Kv1.5-/- with smooth muscle-specific expression Kv1.5 channels) mice were studied following transaortic constriction (TAC). Measurements of left ventricular (LV) fractional shortening (FS) in base and apex, and myocardial blood flow (MBF) were completed with standard and contrast echocardiography. Ribonucleic Acid deep sequencing was performed on LV apex and base from WT and Kv1.5-/- (control and TAC). Changes in gene expression were confirmed by real-time-polymerase chain reaction. MBF was increased with chromonar or by smooth muscle expression of Kv1.5 channels in the TgKv1.5-/-. TAC-induced systolic apical ballooning in Kv1.5-/-, shown as negative FS (P < 0.05 vs. base), which was not observed in WT, Kv1.5-/- with chromonar, or TgKv1.5-/-. Following TAC in Kv1.5-/-, MBF was lower in LV apex than in base. Increasing MBF with either chromonar or in TgKv1.5-/- normalized perfusion and function between LV apex and base (P = NS). Some genetic changes during TTS were reversed by chromonar, suggesting these were independent of TAC and more related to TTS. CONCLUSION: Abnormalities in flow regulation between the LV apex and base cause TTS. When perfusion is normalized between the two regions, normal ventricular function is restored.

Insights into pyrrolysine function from structures of a trimethylamine methyltransferase and its corrinoid protein complex.

The 22nd genetically encoded amino acid, pyrrolysine, plays a unique role in the key step in the growth of methanogens on mono-, di-, and tri-methylamines by activating the methyl group of these substrates for transfer to a corrinoid cofactor. Previous crystal structures of the Methanosarcina barkeri monomethylamine methyltransferase elucidated the structure of pyrrolysine and provide insight into its role in monomethylamine activation. Herein, we report the second structure of a pyrrolysine-containing protein, the M. barkeri trimethylamine methyltransferase MttB, and its structure bound to sulfite, a substrate analog of trimethylamine. We also report the structure of MttB in complex with its cognate corrinoid protein MttC, which specifically receives the methyl group from the pyrrolysine-activated trimethylamine substrate during methanogenesis. Together these structures provide key insights into the role of pyrrolysine in methyl group transfer from trimethylamine to the corrinoid cofactor in MttC.

Validity of dual-energy x-ray absorptiometry for estimation of visceral adipose tissue and visceral adipose tissue change after surgery-induced weight loss in women with severe obesity.

OBJECTIVE: Reliable and simple methods to quantify visceral adipose tissue (VAT) and VAT changes are needed. This study investigated the validity of dual-energy x-ray absorptiometry (DXA) compared with magnetic resonance imaging (MRI) for estimating VAT cross sectionally and longitudinally after surgery-induced weight loss in women with severe obesity. METHODS: Women with obesity (n = 36; mean age 43 [SD 10] years; 89% White) with DXA and MRI before bariatric surgery (T0) at 12 (T12) and 24 months (T24) post surgery were included. CoreScan (GE Healthcare, Chicago, Illinois) estimated VAT from 20% of the distance between the top of the iliac crest and the base of the skull. MRI VAT (total VAT) was measured from the base of the heart to the sacrum/coccyx on a whole-body scan. RESULTS: Mean DXA VAT was 45% of MRI VAT at T0, 46% at T12, and 68% at T24. DXA underestimated change in MRI VAT between T0 and T12 by 26.1% (0.81 kg, p = 0.03) and by 71.7% (0.43 kg, p < 0.001) between T12 and T24. The relationship between DXA VAT and MRI VAT differed between T12 and T24 (p value for interaction = 0.03). CONCLUSIONS: CoreScan lacks validity for comparing VAT across individuals or for estimating the size of changes within individuals; however, within the limits of measurement error, it may provide a useful indicator of whether some VAT change has occurred within an individual.

Mitochondrial DNA integrity and function are critical for endothelium-dependent vasodilation in rats with metabolic syndrome.

Endothelial dysfunction in diabetes is generally attributed to oxidative stress, but this view is challenged by observations showing antioxidants do not eliminate diabetic vasculopathy. As an alternative to oxidative stress-induced dysfunction, we interrogated if impaired mitochondrial function in endothelial cells is central to endothelial dysfunction in the metabolic syndrome. We observed reduced coronary arteriolar vasodilation to the endothelium-dependent dilator, acetylcholine (Ach), in Zucker Obese Fatty rats (ZOF, 34 ± 15% [mean ± standard deviation] 10-3 M) compared to Zucker Lean rats (ZLN, 98 ± 11%). This reduction in dilation occurred concomitantly with mitochondrial DNA (mtDNA) strand lesions and reduced mitochondrial complex activities in the endothelium of ZOF versus ZLN. To demonstrate endothelial dysfunction is linked to impaired mitochondrial function, administration of a cell-permeable, mitochondria-directed endonuclease (mt-tat-EndoIII), to repair oxidatively modified DNA in ZOF, restored mitochondrial function and vasodilation to Ach (94 ± 13%). Conversely, administration of a cell-permeable, mitochondria-directed exonuclease (mt-tat-ExoIII) produced mtDNA strand breaks in ZLN, reduced mitochondrial complex activities and vasodilation to Ach in ZLN (42 ± 16%). To demonstrate that mitochondrial function is central to endothelium-dependent vasodilation, we introduced (via electroporation) liver mitochondria (from ZLN) into the endothelium of a mesenteric vessel from ZOF and restored endothelium-dependent dilation to vasoactive intestinal peptide (VIP at 10-5 M, 4 ± 3% vasodilation before mitochondrial transfer and 48 ± 36% after transfer). Finally, to demonstrate mitochondrial function is key to endothelium-dependent dilation, we administered oligomycin (mitochondrial ATP synthase inhibitor) and observed a reduction in endothelium-dependent dilation. We conclude that mitochondrial function is critical for endothelium-dependent vasodilation.

Reperfusion mediates heme impairment with increased protein cysteine sulfonation of mitochondrial complex III in the post-ischemic heart.

A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSO3H) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c1 in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C122/C125 of cytochrome c1 via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c1. LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSO3H levels at the C236 ligand of the [2Fe2S] cluster of the Rieske iron­sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO3H of the hinge protein at C65 and of cytochrome c1 at C140 and C220, which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSO3H of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSO3H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO3H. In conclusion, reperfusion-mediated heme damage with increased PrSO3H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart.

Resting Energy Expenditure and Organ-Tissue Body Composition 5 Years After Bariatric Surgery.

INTRODUCTION: Bariatric surgery-induced weight loss may reduce resting energy expenditure (REE) and fat-free mass (FFM) disproportionately thereby predisposing patients to weight regain and sarcopenia. METHODS: We compared REE and body composition of African-American and Caucasian Roux-en-Y gastric bypass (RYGB) patients after surgery with a group of non-operated controls (CON). REE by indirect calorimetry; skeletal muscle (SM), trunk organs, and brain volumes by MRI; and FFM by DXA were measured at post-surgery visits and compared with CON (N = 84) using linear regression models that adjusted for relevant covariates. Ns in RYGB were 50, 42, and 30 for anthropometry and 39, 27, 17 for MRI body composition at years 1, 2, and 5 after surgery, respectively. RESULTS: Regression models adjusted for age, weight, height, ethnicity, and sex showed REE differences (RYGB minus CON; mean ± s.e.): year 1 (43.2 ± 34 kcal/day, p = 0.20); year 2 (- 27.9 ± 37.3 kcal/day, p = 0.46); year 5 (114.6 ± 42.3 kcal/day, p = 0.008). Analysis of FFM components showed that RYGB had greater trunk organ mass (~ 0.4 kg) and less SM (~ 1.34 kg) than CON at each visit. REE models adjusted for FFM, SM, trunk organs, and brain mass showed no between-group differences in REE (- 15.9 ± 54.8 kcal/day, p = 0.8; - 46.9 ± 64.9 kcal/day, p = 0.47; 47.7 ± 83.0 kcal/day, p = 0.57, at years 1, 2, and 5, respectively). CONCLUSIONS: Post bariatric surgery patients maintain a larger mass of high-metabolic rate trunk organs than non-operated controls of similar anthropometrics. Interpreting REE changes after weight loss requires an accurate understanding of fat-free mass composition at both the organ and tissue levels. CLINICAL TRIAL REGISTRATION: Long-term Effects of Bariatric Surgery (LABS-2) NCT00465829.

Higher Reliance on Glycolysis Limits Glycolytic Responsiveness in Degenerating Glaucomatous Optic Nerve.

Metabolic dysfunction accompanies neurodegenerative disease and aging. An important step for therapeutic development is a more sophisticated understanding of the source of metabolic dysfunction, as well as to distinguish disease-associated changes from aging effects. We examined mitochondrial function in ex vivo aging and glaucomatous optic nerve using a novel approach, the Seahorse Analyzer. Optic nerves (ON) from the DBA/2J mouse model of glaucoma and the DBA/2-Gpnmb+ control strain were isolated, and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), the discharge of protons from lactate release or byproducts of substrate oxidation, were measured. The glial-specific aconitase inhibitor fluorocitrate was used to limit the contribution of glial mitochondria to OCR and ECAR. We observed significant decreases in maximal respiration, ATP production, and spare capacity with aging. In the presence of fluorocitrate, OCR was higher, with more ATP produced, in glaucoma compared to aged ON. However, glaucoma ON showed lower maximal respiration. In the presence of fluorocitrate and challenged with ATPase inhibition, glaucoma ON was incapable of further upregulation of glycolysis to compensate for the loss of oxidative phosphorylation. Inclusion of 2-deoxyglucose as a substrate during ATPase inhibition indicated a significantly higher proportion of ECAR was derived from TCA cycle substrate oxidation than glycolysis in glaucoma ON. These data indicate that glaucoma axons have limited ability to respond to increased energy demand given their lower maximal respiration and inability to upregulate glycolysis when challenged. The higher ATP output from axonal mitochondria in glaucoma optic nerve compensates for this lack of resiliency but is ultimately inadequate for continued function.

Mitochondrial complex I in the post-ischemic heart: reperfusion-mediated oxidative injury and protein cysteine sulfonation.

A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of O2-. The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to injury. Herein we employed LC-MS/MS to identify protein sulfonation of complex I in mitochondria from the infarct region of rat hearts subjected to 30-min of coronary ligation and 24-h of reperfusion in vivo as well as the mitochondria of sham controls. Mitochondrial preparations from the I/R regions had enhanced sulfonation levels on the cysteine ligands of iron­sulfur clusters, including N3 (C425), N1b (C92), N4 (C226), N2 (C158/C188), and N1a (C134/C139). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus sulfonation of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and an antioxidative function. Increased C134/C139 sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O2- generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C125, C187, C206, C238, C255, C286), 75 kDa (C367, C554, C564, C727), 49 kDa (C146, C326, C347), and PSST (C188). These results were consistent with the consensus indicating that 51 kDa and 75 kDa are two of major subunits hosting regulatory thiols, and their enhanced sulfonation by I/R predisposed the myocardium to further oxidant stress with impaired ubiquinone reduction. MS analysis further showed I/R-mediated enhanced sulfonation at the supernumerary subunits of 42 kDa (C67, C112, C183, C253), 15 kDa (C43), and 13 kDa (C79). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C43 of the 15 kDa subunit forms an intramolecular disulfide bond with C56, which was reported to stabilize complex I structure. C79 of the 13 kDa subunit is involved in Zn2+-binding, which was reported functionally important for complex I assembly. C79 sulfonation by I/R was found to impair Zn2+-binding. No significant enhancement of protein sulfonation was observed in mitochondrial complex I from the rat heart subjected to 30-min ischemia alone in vivo despite a decreased state 3 respiration, suggesting that the physiologic conditions of hyperoxygenation during reperfusion mediated an increase in complex I sulfonation and oxidative injury. In conclusion, sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increasing •O2- production, and mediating redox dysfunction of complex I.

Impairment of pH gradient and membrane potential mediates redox dysfunction in the mitochondria of the post-ischemic heart.

The mitochondrial electrochemical gradient (Δp), which comprises the pH gradient (ΔpH) and the membrane potential (ΔΨ), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide (·O2-) and H2O2 overproduction leading to oxidative injury. However, the role of ΔpH and ΔΨ in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced ·O2- and H2O2 production when compared to state 2 conditions. Oligomycin (increases ΔpH by inhibiting ATP synthase) increased ·O2- and H2O2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial ·O2- and H2O2 generation is dependent on ΔpH and that IR impaired the ΔpH of normal mitochondria. Conversely, nigericin (dissipates ΔpH) dramatically reduced ·O2- and H2O2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced ΔpH impairment in normal mitochondria. Valinomycin, a K+ ionophore used to dissipate ΔΨ, drastically diminished ·O2- and H2O2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that ΔΨ also contributed to ·O2- generation by normal mitochondria and that IR mediated ΔΨ impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by ΔpH restricts electron flow, controls a limited amount of ·O2- generation, and results in a more reduced myocardium; however, IR causes ΔpH impairment and prompts a more oxidized myocardium.

Differential protein acetylation assists import of excess SOD2 into mitochondria and mediates SOD2 aggregation associated with cardiac hypertrophy in the murine SOD2-tg heart.

SOD2 is the primary antioxidant enzyme neutralizing •O2- in mitochondria. Cardiac-specific SOD2 overexpression (SOD2-tg) induces supernormal function and cardiac hypertrophy in the mouse heart. However, the reductive stress imposed by SOD2 overexpression results in protein aggregation of SOD2 pentamers and differential hyperacetylation of SOD2 in the mitochondria and cytosol. Here, we studied SOD2 acetylation in SOD2-tg and wild-type mouse hearts. LC-MS/MS analysis indicated the presence of four acetylated lysines in matrix SOD2 and nine acetylated lysines in cytosolic SOD2 from the SOD2-tg heart. However, only one specific acetylated lysine residue was detected in the mitochondria of the wild-type heart, which was consistent with Sirt3 downregulation in the SOD2-tg heart. LC-MS/MS further detected hyperacetylated SOD2 with a signaling peptide in the mitochondrial inner membrane and matrix of the SOD2-tg heart, indicating partial arrest of the SOD2 precursor in the membrane during translocation into the mitochondria. Upregulation of HSP 70 and cytosolic HSP 60 enabled the translocation of excess SOD2 into mitochondria. In vitro acetylation of matrix SOD2 with Ac2O deaggregated pentameric SOD2, restored the profile of cytosolic SOD2 hyperacetylation, and decreased matrix SOD2 activity. As revealed by 3D structure, acetylation of K89, K134, and K154 of cytosolic SOD2 induces unfolding of the tertiary structure and breaking of the salt bridges that are important for the quaternary structure, suggesting that hyperacetylation and HSP 70 upregulation maintain the unfolded status of SOD2 in the cytosol and mediate the import of SOD2 across the membrane. Downregulation of Sirt3, HSP 60, and presequence protease in the mitochondria of the SOD2-tg heart promoted protein misfolding that led to pentameric aggregation.

Impaired coronary metabolic dilation in the metabolic syndrome is linked to mitochondrial dysfunction and mitochondrial DNA damage.

Mitochondrial dysfunction in obesity and diabetes can be caused by excessive production of free radicals, which can damage mitochondrial DNA. Because mitochondrial DNA plays a key role in the production of ATP necessary for cardiac work, we hypothesized that mitochondrial dysfunction, induced by mitochondrial DNA damage, uncouples coronary blood flow from cardiac work. Myocardial blood flow (contrast echocardiography) was measured in Zucker lean (ZLN) and obese fatty (ZOF) rats during increased cardiac metabolism (product of heart rate and arterial pressure, i.v. norepinephrine). In ZLN increased metabolism augmented coronary blood flow, but in ZOF metabolic hyperemia was attenuated. Mitochondrial respiration was impaired and ROS production was greater in ZOF than ZLN. These were associated with mitochondrial DNA (mtDNA) damage in ZOF. To determine if coronary metabolic dilation, the hyperemic response induced by heightened cardiac metabolism, is linked to mitochondrial function we introduced recombinant proteins (intravenously or intraperitoneally) in ZLN and ZOF to fragment or repair mtDNA, respectively. Repair of mtDNA damage restored mitochondrial function and metabolic dilation, and reduced ROS production in ZOF; whereas induction of mtDNA damage in ZLN reduced mitochondrial function, increased ROS production, and attenuated metabolic dilation. Adequate metabolic dilation was also associated with the extracellular release of ADP, ATP, and H2O2 by cardiac myocytes; whereas myocytes from rats with impaired dilation released only H2O2. In conclusion, our results suggest that mitochondrial function plays a seminal role in connecting myocardial blood flow to metabolism, and integrity of mtDNA is central to this process.

Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction.

We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H2O2 contributes to CBF regulation; however, excessive H2O2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H2O2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H2O2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H2O2-induced vasodilation was significantly inhibited in db/db and V1KO mice. H2O2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H2O2-induced vasodilation occurs, in part, via TRPV1. Acute H2O2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H2O2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H2O2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H2O2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H2O2 exposure attenuated TRPV1 currents. Verification of H2O2-mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H2O2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

Overexpressing superoxide dismutase 2 induces a supernormal cardiac function by enhancing redox-dependent mitochondrial function and metabolic dilation.

During heightened cardiac work, O2 consumption by the heart benefits energy production via mitochondria. However, some electrons leak from the respiratory chain and yield superoxide, which is rapidly metabolized into H2O2 by SOD2. To understand the systemic effects of the metabolic dilator, H2O2, we studied mice with cardiac-specific SOD2 overexpression (SOD2-tg), which increases the H2O2 produced by cardiac mitochondria. Contrast echocardiography was employed to evaluate cardiac function, indicating that SOD2-tg had a significantly greater ejection fraction and a lower mean arterial pressure (MAP) that was partially normalized by intravenous injection of catalase. Norepinephrine-mediated myocardial blood flow (MBF) was significantly enhanced in SOD2-tg mice. Coupling of MBF to the double product (Heart Rate×MAP) was increased in SOD2-tg mice, indicating that the metabolic dilator, "spilled" over, inducing systemic vasodilation. The hypothesis that SOD2 overexpression effectively enhances mitochondrial function was further evaluated. Mitochondria of SOD2-tg mice had a decreased state 3 oxygen consumption rate, but maintained the same ATP production flux under the basal and L-NAME treatment conditions, indicating a higher bioenergetic efficiency. SOD2-tg mitochondria produced less superoxide, and had lower redox activity in converting cyclic hydroxylamine to stable nitroxide, and a lower GSSG concentration. EPR analysis of the isolated mitochondria showed a significant decrease in semiquinones at the SOD2-tg Qi site. These results support a more reductive physiological setting in the SOD2-tg murine heart. Cardiac mitochondria exhibited no significant differences in the respiratory control index between WT and SOD2-tg. We conclude that SOD2 overexpression in myocytes enhances mitochondrial function and metabolic vasodilation, leading to a phenotype of supernormal cardiac function.

Increased mitochondrial prooxidant activity mediates up-regulation of Complex I S-glutathionylation via protein thiyl radical in the murine heart of eNOS(-/-).

In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS(•)) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 and 75-kDa subunits was detected under the conditions of (•)O2(-) production, and mass spectrometry confirmed that formation of Complex I PrS(•) mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS(•) (Kang et al., Free Radical Biol. Med.52:962-973; 2012). In this investigation, we tested our hypothesis in the murine heart of eNOS(-/-). The eNOS(-/-) mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS(-/-) myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in (•)O2(-) generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS(-/-) murine heart. When Complex I from the mitochondria of the eNOS(-/-) murine heart was analyzed by immunospin trapping and probed with anti-GSH antibody, both PrS(•) and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS(•), supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS(•) is a unique pathway for the redox regulation of mitochondrial function in vivo.

Continued loss in visceral and intermuscular adipose tissue in weight-stable women following bariatric surgery.

OBJECTIVE: To assess changes in total (TAT), subcutaneous (SAT), visceral (VAT), and intermuscular (IMAT) adipose tissue by whole-body MRI before surgery and at 12 months and 24 months post-surgery in a subset of participants of the Longitudinal Assessment of Bariatric Surgery-2. METHODS: From 0 to 12 months, n = 20 females and 3 males; from 12 to 24 months, n = 42 females and 7 males. Paired t-tests and GLM repeated measures examined changes in TAT, SAT, VAT, and IMAT at 12 and 24 months, with sex and age as covariates. RESULTS: Changes from 0 to 12 months included weight (-41.9 ± 12.1 kg; -36%), TAT (-33.5 ± 9.6 kg; -56%), SAT (-29.2 ± 8.2 kg; -55%), VAT (-3.3 ± 1.6 kg; -73%), and IMAT (-0.99 ± 0.68 kg; -50%), all P < 0.001. In females, from 12 to 24 months, despite relative weight stability (-1.8 ± 6.5 kg, -2%; P = 0.085), VAT (-0.5 ± 0.7 kg; -30%; P < 0.001) and IMAT (-0.2 ± 0.4 kg; -14%; P = 0.012) decreased further. In males, from 12 to 24 months, weight increased (5.1 ± 5.2 kg; 6%; P = 0.04) with no significant changes in TAT or sub-depots. CONCLUSIONS: Bariatric surgery continues to induce favorable changes in body composition, i.e., persistent adipose tissue loss at 24 months in the absence of further significant weight loss.

BCNU-induced gR2 defect mediates S-glutathionylation of Complex I and respiratory uncoupling in myocardium.

A deficiency of mitochondrial glutathione reductase (or GR2) is capable of adversely affecting the reduction of GSSG and increasing mitochondrial oxidative stress. BCNU [1,3-bis (2-chloroethyl)-1-nitrosourea] is an anticancer agent and known inhibitor of cytosolic GR ex vivo and in vivo. Here we tested the hypothesis that a BCNU-induced GR2 defect contributes to mitochondrial dysfunction and subsequent impairment of heart function. Intraperitoneal administration of BCNU (40 mg/kg) specifically inhibited GR2 activity by 79.8 ± 2.7% in the mitochondria of rat heart. However, BCNU treatment modestly enhanced the activities of mitochondrial Complex I and other ETC components. The cardiac function of BCNU-treated rats was analyzed by echocardiography, revealing a systolic dysfunction associated with decreased ejection fraction, decreased cardiac output, and an increase in left ventricular internal dimension and left ventricular volume in systole. The respiratory control index of isolated mitochondria from the myocardium was moderately decreased after BCNU treatment, whereas NADH-linked uncoupling of oxygen consumption was significantly enhanced. Extracellular flux analysis to measure the fatty acid oxidation of myocytes indicated a 20% enhancement after BCNU treatment. When the mitochondria were immunoblotted with antibodies against GSH and UCP3, both protein S-glutathionylation of Complex I and expression of UCP3 were significantly up-regulated. Overexpression of SOD2 in the myocardium significantly reversed BCNU-induced GR2 inhibition and mitochondrial impairment. In conclusion, BCNU-mediated cardiotoxicity is characterized by the GR2 deficiency that negatively regulates heart function by impairing mitochondrial integrity, increasing oxidative stress with Complex I S-glutathionylation, and enhancing uncoupling of mitochondrial respiration.

Oxidative modifications of mitochondria complex II.

Increased superoxide (O2 (·-)) and nitric oxide (NO) production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In the complex II, oxidative impairment, decreased protein S-glutathionylation, and increased protein tyrosine nitration at the 70 kDa subunit occur in the post-ischemic myocardium (Zhang et al., Biochemistry 49:2529-2539, 2010; Chen et al., J Biol Chem 283:27991-28003, 2008; Chen et al., J Biol Chem 282: 32640-32654, 2007). To gain the deeper insights into ROS-mediated oxidative modifications relevant in myocardial infarction, isolated complex II is subjected to in vitro oxidative modifications with GSSG (to induce cysteine S-glutathionylation) or OONO(-) (to induce tyrosine nitration). Here, we describe the protocol to characterize the specific oxidative modifications at the 70 kDa subunit by nano-LC/MS/MS analysis. We further demonstrate the cellular oxidative modification with protein nitration/S-glutathionylation with immunofluorescence microscopy using the antibodies against 3-nitrotyrosine/glutathione and complex II 70 kDa polypeptide (AbGSC90) in myocytes under conditions of oxidative stress.

Protein thiyl radical mediates S-glutathionylation of complex I.

Complex I is a critical site of O(2)(•-) production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, complex I protein thiols at the 51- and 75-kDa subunits are reversibly S-glutathionylated. The mechanism of complex I S-glutathionylation is mainly obtained from insight into GSSG-mediated thiol-disulfide exchange, which would require a dramatic decline in the GSH/GSSG ratio. Intrinsic complex I S-glutathionylation can be detected in the rat heart at a relatively high GSH/GSSG ratio (J. Chen et al., J. Biol. Chem. 285:3168-3180, 2010). Thus, we hypothesized that reactive thiyl radical is more likely to mediate protein S-glutathionylation of complex I. Here we employed immuno-spin trapping and tandem mass spectrometry (LC/MS/MS) to test the hypothesis in the 75-kDa subunit from S-glutathionylated complex I. Under the conditions of O(2)(•-) production in the presence of GSH, we detected complex I S-glutathionylation at Cys-226, Cys-367, and Cys-727 of the 75-kDa subunit. Addition of a radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), significantly decreased complex I S-glutathionylation and subsequently increased the protein radical adduct of complex I-DMPO as detected by immunoblotting using an anti-DMPO antibody. LC/MS/MS analysis indicated that Cys-226, Cys-554, and Cys-727 were involved in DMPO binding, confirming that formation of the complex I thiyl radical mediates S-glutathionylation. LC/MS/MS analysis also showed that Cys-554 and Cys-727 were S-sulfonated under conditions of O(2)(•-) generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O(2)(•-) generation, complex I protein radical and S-glutathionylation were increased. Thus mediation of complex I S-glutathionylation by the protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function.

What is the best way to contour lung tumors on PET scans? Multiobserver validation of a gradient-based method using a NSCLC digital PET phantom.

PURPOSE: To evaluate the accuracy and consistency of a gradient-based positron emission tomography (PET) segmentation method, GRADIENT, compared with manual (MANUAL) and constant threshold (THRESHOLD) methods. METHODS AND MATERIALS: Contouring accuracy was evaluated with sphere phantoms and clinically realistic Monte Carlo PET phantoms of the thorax. The sphere phantoms were 10-37 mm in diameter and were acquired at five institutions emulating clinical conditions. One institution also acquired a sphere phantom with multiple source-to-background ratios of 2:1, 5:1, 10:1, 20:1, and 70:1. One observer segmented (contoured) each sphere with GRADIENT and THRESHOLD from 25% to 50% at 5% increments. Subsequently, seven physicians segmented 31 lesions (7-264 mL) from 25 digital thorax phantoms using GRADIENT, THRESHOLD, and MANUAL. RESULTS: For spheres <20 mm in diameter, GRADIENT was the most accurate with a mean absolute % error in diameter of 8.15% (10.2% SD) compared with 49.2% (51.1% SD) for 45% THRESHOLD (p < 0.005). For larger spheres, the methods were statistically equivalent. For varying source-to-background ratios, GRADIENT was the most accurate for spheres >20 mm (p < 0.065) and <20 mm (p < 0.015). For digital thorax phantoms, GRADIENT was the most accurate (p < 0.01), with a mean absolute % error in volume of 10.99% (11.9% SD), followed by 25% THRESHOLD at 17.5% (29.4% SD), and MANUAL at 19.5% (17.2% SD). GRADIENT had the least systematic bias, with a mean % error in volume of -0.05% (16.2% SD) compared with 25% THRESHOLD at -2.1% (34.2% SD) and MANUAL at -16.3% (20.2% SD; p value <0.01). Interobserver variability was reduced using GRADIENT compared with both 25% THRESHOLD and MANUAL (p value <0.01, Levene's test). CONCLUSION: GRADIENT was the most accurate and consistent technique for target volume contouring. GRADIENT was also the most robust for varying imaging conditions. GRADIENT has the potential to play an important role for tumor delineation in radiation therapy planning and response assessment.