Thioredoxin-1 maintains mechanistic target of rapamycin (mTOR) function during oxidative stress in cardiomyocytes

Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. As such, Trx1 helps protect the heart against stresses, such as ischemia and pressure overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, and survival. We have shown previously that mTOR activity is increased in response to myocardial ischemia-reperfusion injury. However, whether Trx1 interacts with mTOR to preserve heart function remains unknown. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we show here that mTOR is a direct interacting partner of Trx1 in the heart. In response to H2O2 treatment in cardiomyocytes, mTOR exhibited a high molecular weight shift in non-reducing SDS-PAGE in a 2-mercaptoethanol-sensitive manner, suggesting that mTOR is oxidized and forms disulfide bonds with itself or other proteins. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. Immune complex kinase assays disclosed that H2O2 treatment diminished mTOR kinase activity, indicating that mTOR is inhibited by oxidation. Of note, Trx1 overexpression attenuated both H2O2-mediated mTOR oxidation and inhibition, whereas Trx1 knockdown increased mTOR oxidation and inhibition. Moreover, Trx1 normalized H2O2-induced down-regulation of metabolic genes and stimulation of cell death, and an mTOR inhibitor abolished Trx1-mediated rescue of gene expression. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine. These results suggest that Trx1 protects cardiomyocytes against stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death

Matrix-assisted polymer pen lithography induced Staudinger Ligation

The Staudinger Ligation has been combined with Polymer Pen Lithography to create patterns of fluorescent and redox-active inks with 1-micrometer scale feature diameters over centimeter-scale areas. This report presents a straightforward strategy to expand the scope of organic reactions employed in surface science

Comparison of Pharmacy Refill Data With Chemical Adherence Testing in Assessing Medication Nonadherence in a Safety Net Hospital Setting

Background Pharmacy fill data are a practical tool for assessing medication nonadherence. However, previous studies have not compared the accuracy of pharmacy fill data to measurement of plasma drug levels, or chemical adherence testing (CAT). Methods and Results We performed a cross‐sectional study in patients with uncontrolled hypertension in outpatient clinics in a safety net health system. Plasma samples were obtained for measurement of common cardiovascular drugs, including calcium channel blockers, thiazide diuretics, beta blockers, angiotensin‐converting enzyme inhibitors/angiotensin receptor blockers, and statins, using liquid chromatography mass spectrometry. Proportion of days covered (PDC), a method for tracking pharmacy fill data, was calculated via linkages with Surescripts, and its diagnostic test characteristics were compared with CAT. Among 77 patients with uncontrolled hypertension, 13 (17%) were nonadherent to at least 1 antihypertensive drug and 23 (37%) were nonadherent to statins by CAT. PDC was significantly lower in the nonadherent versus the adherent group by CAT only among patients prescribed an angiotensin‐converting enzyme inhibitor/angiotensin receptor blocker or statin (all P<0.05) but not in patients prescribed other drug classes. The sensitivity and specificity of PDC in detecting nonadherence to angiotensin‐converting enzyme inhibitors/angiotensin receptor blockers and statin drugs by CAT were 75% to 82% and 56% to 79%, respectively. The positive predictive value of PDC in detecting nonadherence was only 11% to 27% for antihypertensive drugs and 45% for statins. Conclusions PDC is useful in detecting nonadherence to angiotensin‐converting enzyme inhibitors/angiotensin receptor blockers and statins but has limited usefulness in detecting nonadherence to calcium channel blockers, beta blockers, or thiazide diuretics and has a low positive predictive value for all drug classes

An Ideal PPAR Response Element Bound to and Activated by PPARα

<div><p>Peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor, plays an important role in the transcription of genes involved in fatty acid metabolism through heterodimerization with the retinoid x receptor (RXR). The consensus sequence of the PPAR response element (PPRE) is composed of two AGGTCA-like sequences directionally aligned with a single nucleotide spacer. PPARα and RXR bind to the 5’ and 3’ hexad sequences, respectively. However, the precise sequence definition of the PPRE remains obscure, and thus, the consensus sequence currently available remains AGGTCANAGGTCA with unknown redundancy. The vague PPRE sequence definition poses an obstacle to understanding how PPARα regulates fatty acid metabolism. Here we show that, rather than the generally accepted 6-bp sequence, PPARα actually recognized a 12-bp DNA sequence, of which the preferred binding sequence was WAWVTRGGBBAH. Additionally, the optimized RXRα hexad binding sequence was RGKTYA. Thus, the optimal PPARα/RXRα heterodimer binding sequence was WAWVTRGGBBAHRGKTYA. The single nucleotide substitution, which reduces binding of RXRα to DNA, attenuated PPARα-induced transcriptional activation, but this is not always true for PPARα. Using the definition of the PPRE sequence, novel PPREs were successfully identified. Taken altogether, the provided PPRE sequence definition contributes to the understanding of PPARα signaling by identifying PPARα direct target genes with functional PPARα response elements.</p></div

The 3’ core hexad sequence for RXRα binding.

<p>(A) Tandem AGGTCA sequences are required for detecting RXRα DNA binding. The indicated biotin-labeled DNA was incubated with recombinant RXRα. (B) Determination of the optimal DNA sequences for RXRα binding. Biotin-labeled double-stranded oligonucleotides comprising DR1 shown in Fig 6A were incubated with recombinant RXRα and unlabeled competitors (10- and 30-fold excess of biotin-labeled DNA). The competitors had all 4 possible nucleotides at the indicated positions (+1 to +6). All signals without competitors are identical among the four panels at each position. A dotted line indicates a discontinued signal but they are derived from identical blots/membranes. (C-D) The effect of non-preferred nucleotides in the 3’ core hexad sequence for RXRα binding on PPARα-induced transcriptional activation. Reporter gene assays were performed with the indicated sequences of PPRE (n = 6–9).</p

Identification of novel PPARα response elements using the determined sequence definition.

<p>(A) The optimized binding sequence for PPARα and RXRα (top). The densitometric analysis of the Western blot analyses (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134996#pone.0134996.g003" target="_blank">3</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134996#pone.0134996.g006" target="_blank">6</a>) is shown by heat map (bottom). Signal densities were measured with the ImageJ program. The signal derived from a non-competitor was defined as 100. The optimized binding sequence was defined by the nucleotide sequences representing more than 80% competitive inhibition of the PPRE sequence we used (AAATGT-AGGTCA-A-AGGTCA). (B) Schematic representation of the reporter constructs driven by the promoters of the indicated genes. The potential PPRE is indicated by red arrows. (C) Reporter gene activation by PPARα. (D) Single nucleotide substitution at the +3 position of the 3’ hexad element of the putative PPRE in the <i>Acot2</i> and <i>Fbp2</i> promoters abolished PPARα-induced promoter activation. (C-D) The indicated reporter gene constructs were transfected into cells. Reporter assays were performed (n = 5–18).</p

The putative PPARα response element in the heart.

<p>(A-B) PPARα is a major PPAR isoform in the heart. Relative copy numbers of PPAR isoforms at the mRNA level in FVB background mice (A) and in primary cultured neonatal cardiac myocytes (B) were examined by qPCR with isoform-specific PCR primers (n = 12 (A) and 5 to 6 (B)). (C) PPARα is required for cardiac systolic function at baseline. Echocardiographic measurements were performed on the indicated mouse genotypes. The cardiac systolic function was evaluated by determination of fractional shortening (n = 16–19). (D) PPARα is required for cardiac fatty acid oxidation activity. Fatty acid oxidation activity was measured in PPARα knockout mice (n = 5–9). (E) PPAR target gene expression in PPARα knockout mice. The relative mRNA levels were examined by qPCR. Both sexes of 2- to 6-month-old mice were used. The numbers of mice examined in each experimental group were indicated. (F) Relative PPAR target gene expression in Tg-PPARα mice. The heat map of PPAR target genes was generated according to the microarray results for NTg and Tg-PPARα. (G) Intrinsic PPRE sequences harbored by PPAR target genes upregulated in Tg-PPARα. The arrow indicates the transcription start site and direction of the gene body. The PPRE sequences were taken from published papers. (H) Putative PPRE consensus sequence in the heart. The PPRE sequence was generated using the PPREs shown in (G) with WebLogo [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134996#pone.0134996.ref026" target="_blank">26</a>].</p

Determination of the 5’ extended sequence of the PPRE.

<p>(A) The DNA sequence of the 5’ extended region of DR1 is critical for PPARα binding. Biotin-labeled DNAs comprised of DR1 with the most and least frequent sequences in the 5’ extended region shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134996#pone.0134996.g001" target="_blank">Fig 1G</a> were incubated with recombinant PPARα. PPARα bound to DNA was detected by Western blot analyses. (B) The 5’ extended sequence of DR1 is critical for PPARα ligand-induced transcriptional activation. Luciferase reporter genes driven by the PPREs/DR1s with the most and least frequent 6-bp sequences of the 5’extended region were stimulated with WY14643. (C) PPARα recognizes 5 bp of the 5’ extended region of the 5’ core hexad element. The competitors have less frequent nucleotides from positions -6 to -1. (D) PPARα does not recognize the 5’ extended region from position -7. The competitors have the indicated DNA sequences from -7 to -11. (E) Determination of the optimized PPARα binding sequence in the 5’ extended region. The competitors have all 4 possible nucleotides from positions -6 to -1. (C-E) The competition assays of PPARα binding were performed with biotin-labeled DNA and unlabeled competitors (10- and 30-fold excess of biotin-labeled DNA). (C- E) All signals without competitors are identical among the 4 panels at each position. A dotted line indicates discontinued signal but they are derived from identical blot/membrane. (F-G) The effect of a non-preferred nucleotide in the 5’ extended region on PPARα-induced transcriptional activation. Reporter gene assays were performed with the indicated PPRE sequences (n = 5–9).</p

PPRE sequence differentially directs transcriptional activation by liganded and unliganded PPARα.

<p>(A) Forced expression of PPARα preferentially activates the major direction of the PPRE in primary cultured myocytes. (B) Overexpression of PPARα-induced transcriptional activation was enhanced with the PPRE containing a G nucleotide spacer in primary cultured myocytes. (C) Single nucleotide substitutions that reduce PPARα binding at positions +4 to +6 did not significantly affect PPARα-overexpression-induced reporter activity in primary cultured myocytes. (D-E) The effect of PPRE orientation in the ligand (D) and PPARα-overexpression (E)-induced transcriptional activation in Cos7 cells. (F-G) The effect of G nucleotide spacer in the ligand- (F) and PPARα-overexpression (G)-induced transcriptional activation in Cos7 cells. (H-I) The effect of nucleotide substitution that reduced PPARα DNA binding in the ligand (H) and PPARα-overexpression (I)-induced transcriptional activation in Cos7 cells. Reporter gene assays were performed with the indicated sequences of PPRE (n = 5–6).</p