Physiological role for S-nitrosylation of RyR1 in skeletal muscle function and development.

Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca release from the sarcoplasmic reticulum through the ryanodine receptor/Ca-release channel RyR1. The RyR1 contains ∼100 Cys thiols of which ∼30 comprise an allosteric network subject to posttranslational modification by S-nitrosylation, S-palmitoylation and S-oxidation. However, the role and function of these modifications is not understood.

The value of MRI-based radiomics for evaluating early parotid gland injury in primary Sjögren's syndrome.

Objective: This study aimed to evaluate the value of machine learning models (ML) based on MRI radiomics in diagnosing early parotid gland injury in primary Sjögren's syndrome (pSS).

Methods: A total of 164 patients (114 in the training cohort and 50 in the testing cohort) with pSS (n=82) or healthy controls (HC) (n=82) were enrolled. Itksnap software was used to perform two-dimensional segmentation of the bilateral parotid glands on T1-weighted (T1WI) and fat-suppressed T2-weighted imaging (fs-T2WI) images.

Oxidative stress, NADPH oxidases, and arteries.

Atherosclerosis and its major complications - myocardial infarction and stroke - remain major causes of death and disability in the United States and world-wide. Indeed, with dramatic increases in obesity and diabetes mellitus, the prevalence and public health impact of cardiovascular diseases (CVD) will likely remain high. Major advances have been made in development of new therapies to reduce the incidence of atherosclerosis and CVD, in particular for treatment of hypercholesterolemia and hypertension.

Regulation of the skeletal muscle ryanodine receptor/Ca2+-release channel RyR1 by S-palmitoylation.

The ryanodine receptor/Ca(2+)-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca(2+) release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation.

Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor/Ca2+ release channel (RyR1): sites and nature of oxidative modification.

In mammalian skeletal muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca(2+)-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO2) through O2-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O2) versus low pO2 (1% O2), but their identity among the 100 Cys residues/RyR1 monomer is unknown.

Aberrant S-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart.

Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca(2+) handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca(2+) leak/sparks in isolated myocytes, likely dependent on the underlying physiological state. It remains unknown, however, whether such molecular aberrancies are causally related to arrhythmogenesis in the intact heart.

Off-target thiol alkylation by the NADPH oxidase inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870).

Specific inhibitors of the production of reactive oxygen species (ROS) by the NADPH oxidases (Nox's) are potentially important therapeutic agents in the wide range of human diseases that are characterized by excessive ROS production. It has been proposed that VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3- triazolo[4,5-d]pyrimidine), identified as an inhibitor of Nox2 by small-molecule screening, may serve as an example of such an agent. Here we show that VAS2870 inhibits ROS production in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle, previously identified with Nox4, and thereby abrogates O(2)-coupled redox regulation of the ryanodine receptor-Ca(2+) channel (RyR1).

Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function.

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and β-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed β-adrenergic inotropic responses that were associated with impaired β-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak.

Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4.

Physiological sensing of O(2) tension (partial O(2) pressure, pO(2)) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO(2) to altered calcium release through the ryanodine receptor-Ca(2+)-release channel (RyR1). Reactive oxygen species are generated in proportion to pO(2) by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca(2+) release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle.

S-Nitrosylation of cardiac ion channels.

Nitric oxide (NO) exerts ubiquitous signaling via posttranslational modification of cysteine residues, a reaction termed S-nitrosylation. Important substrates of S-nitrosylation that influence cardiac function include receptors, enzymes, ion channels, transcription factors, and structural proteins. Cardiac ion channels subserving excitation-contraction coupling are potentially regulated by S-nitrosylation.

Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking.

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia coli-expressed glutathione-S-transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection.

Adeno-associated virus-mediated ILK gene silencing in the rat NAc core.

In this study we established conditional silencing of integrin-linked kinase (ILK) expression in Sprague-Dawley rat brain by microinjection of rAAV-2-carrying, Tet-On-regulated siRNA expression cassette into nucleus accumbens (NAc) core and induction with doxycycline. We demonstrated that inhibition of ILK expression was effectively induced by administration of doxycycline for 2 weeks while ILK expression was restored after withdrawing doxycycline for 8 days. Increases in GFAP and OX42 expression were observed 5 weeks post virus injection.

A SNO storm in skeletal muscle.

Dysregulated S-nitrosylation of proteins characterizes a broad array of human disorders, but its role in disease etiology is not well understood. Two new studies (Durham et al., 2008; Bellinger et al.

Reversal of cocaine sensitization-induced behavioral sensitization normalizes GAD67 and GABAA receptor alpha2 subunit expression, and PKC zeta activity.

We have recently shown in rats that cocaine-induced behavioral sensitization can be reversed by a 5-day treatment with ondansetron given 3.5 h after daily pergolide injections. In this study we further investigated the molecular/neurochemical alterations underlying cocaine sensitization and pergolide/ondansetron-mediated reversal.

Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase.

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme.

Mammalian selenoprotein thioredoxin-glutathione reductase. Roles in disulfide bond formation and sperm maturation.

Thioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module.

Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide.

It is generally accepted that inhibition of nitric oxide synthase (NOS) facilitates, and thus nitric oxide (NO) inhibits, contractility of skeletal muscle. However, standard assessments of contractility are carried out at a nonphysiological oxygen tension [partial pressure of oxygen (pO2)] that can interfere with NO signaling (95% O2). We therefore examined, in normal and neuronal NOS (nNOS)-deficient mice, the influence of pO2 on whole-muscle contractility and on myocyte calcium flux and sarcomere shortening.