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Substance use disorders are defined by persistent drug consumption despite adverse consequences. Accordingly, we developed two fentanyl-vs-shock avoidance/escape choice procedures in which male and female rats responded under a fixed-ratio (FR)1:FR1 concurrent schedule of shock avoidance/escape and IV fentanyl under either mutually exclusive or non-exclusive choice conditions. Initial experiments using a discrete-trial procedure determined behavioral allocation between mutually exclusive shock avoidance/escape and different fentanyl doses (0.32-18 μg/kg/infusion; Experiment 1). Shock intensity (0.1-0.7 mA) and shock avoidance/escape response requirement (FR1-16) were also manipulated (Experiment 2). Next, we used a free-operant procedure in which shock avoidance/escape and fentanyl were continuously available under non-exclusive conditions, and response-shock (R-S) interval (30-1000 s) was manipulated (Experiment 3). Finally, we tested the hypothesis that extended-access fentanyl self-administration would produce fentanyl dependence, establish fentanyl withdrawal as an endogenous negative reinforcer, and increase fentanyl choice in both procedures (Experiments 4 and 5). The shock avoidance/escape contingency decreased fentanyl self-administration, and rats consistently chose shock avoidance/escape over fentanyl in both choice conditions. Decreasing shock intensity or increasing shock avoidance/escape response requirement failed to increase fentanyl choice, suggesting that fentanyl and shock avoidance/escape are independent economic commodities. Increasing the R-S interval increased fentanyl choice but failed to increase shock delivery. Extended fentanyl access engendered high fentanyl intake and opioid withdrawal signs but failed to increase fentanyl choice under either choice condition. These results suggest that neither positive fentanyl reinforcement nor negative reinforcement by fentanyl withdrawal is sufficient to reduce shock avoidance/escape-maintained responding and increase foot shock as an adverse consequence., (© 2024. The Author(s).)

Background: The emergence and growth in antibiotic resistant bacteria is a critical public health problem exacerbated by the misuse of antibiotics. Children frequently succumb to illness and are often treated with antibiotic medicines which may be used improperly by the parent. There is limited evidence of the factors influencing parental decision-making about the use of antibiotics in low-resource contexts. The aim of this systematic review was to understand and describe how parents living in rural and remote locations make choices about their children's antibiotic use., Method: The CINAHL, Web of Science, Medline, Scopus and Academic Search Premier databases were systematically searched from 31 January until 28 June in 2023. No date restrictions were applied and additional search methods were utilised to identify further studies that met inclusion criteria. Eligibility criteria included studies which reported on factors contributing to parental decisions about their children's use of antibiotics in rural and remote settings. The Joanna Briggs Institute Critical Appraisal Checklists were employed to evaluate studies. Characteristics and findings were extracted from studies, and data was synthesised descriptively and presented in summary tables., Results: A total of 3827 articles were screened and 25 worldwide studies comprising of quantitative, qualitative and prospective designs were included in the review. Studies that reported the number of rural caregivers consisted of 12 143 participants. Data analysis produced six broad themes representing the mechanisms that influenced parents in their access and use of antibiotics: the child's symptoms; external advice and influences; parent-related determinants; barriers to healthcare; access to antibiotics; and socio-demographic characteristics., Conclusions: A number of factors that influence parents' prudent use of antibiotics in rural contexts were identified. In seeking to enhance appropriate use of antibiotics by parents in rural and remote settings, these determinants can serve to inform interventions. However, the identified studies all relied upon parental self-reports and not all studies reviewed reported survey validation. Further research incorporating validated measures and intervention strategies is required., Registration Details: Should my child be given antibiotics? A systematic review of parental decision making in rural and remote locations; CRD42023382169; 29 January 2023 (date of registration). Available from PROSPERO., (© 2024. The Author(s).)

Introduction: Intermediate efficacy mu opioid receptor (MOR) agonists have potential to retain analgesic effectiveness while improving safety, but the optimal MOR efficacy for effective and safe opioid analgesia is unknown. Preclinical assays of pain-depressed behavior can assess effects of opioids and other candidate analgesics on pain-related behavioral depression, which is a common manifestation of clinically relevant pain and target of pain treatment. Accordingly, the present study goal was to validate a novel assay of pain-depressed locomotor behavior in mice and evaluate the role of MOR efficacy as a determinant of opioid analgesic effects and related safety measures., Methods: Male and female ICR mice were tested in a locomotor chamber consisting of 2 compartments connected by a doorway that contained a 1-inch-tall barrier. Dependent measures during 15-min behavioral sessions included crosses between compartments (which required vertical activity to surmount the barrier) and total movement counts (which required horizontal activity to break photobeams in each compartment)., Results and Discussion: Intraperitoneal injection of lactic acid (IP acid) produced a concentration- and time-dependent depression of both endpoints. Optimal blockade of IP acid-induced behavioral depression with minimal motor impairment was achieved with intermediate-efficacy MOR treatments that also produced less gastrointestinal-transit inhibition and respiratory depression than the high-efficacy MOR agonist fentanyl. Sex differences in treatment effects were rare. Overall, these findings validate a novel procedure for evaluating opioids and other candidate analgesic effects on pain-related behavioral depression in mice and support continued research with intermediate-efficacy MOR agonists as a strategy to retain opioid analgesic effectiveness with improved safety., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Negus, Akbarali, Kang, Lee, Marsh, Santos and Zhang.)

Low-efficacy mu-opioid receptor (MOR) agonists represent promising therapeutics, but existing compounds (e.g., buprenorphine, nalbuphine) span a limited range of low MOR efficacies and have poor MOR selectivity. Accordingly, new and selective low-efficacy MOR agonists are of interest. A novel set of chiral C9-substituted phenylmorphans has been reported to display improved MOR selectivity and a range of high-to-low MOR efficacies under other conditions; however, a full opioid receptor binding profile for these drugs has not been described. Additionally, studies in mice will be useful for preclinical characterization of these novel compounds, but the pharmacology of these drugs in mice has also not been examined. Accordingly, the present study characterized the binding selectivity and in vitro efficacy of these compounds using assays of opioid receptor binding and ligand-stimulated [ 35 S]GTPɣS binding. Additionally, locomotor effects were evaluated as a first step for in vivo behavioral assessment in mice. The high-efficacy MOR agonist and clinically effective antidepressant tianeptine was included as a comparator. In binding studies, all phenylmorphans showed improved MOR selectivity relative to existing lower-efficacy MOR agonists. In the ligand-stimulated [ 35 S]GTPɣS binding assay, seven phenylmorphans had graded levels of sub-buprenorphine MOR efficacy. In locomotor studies, the compounds again showed graded efficacy with a rapid onset and ≥1 h duration of effects, evidence for MOR mediation, and minor sex differences. Tianeptine functioned as a high-efficacy MOR agonist. Overall, these in vitro and in vivo studies support the characterization of these compounds as MOR-selective ligands with graded MOR efficacy and utility for further behavioral studies in mice., (© 2023 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Background: Inappropriate use of antibiotics can promote bacterial resistance, which is a growing public health concern. As children are amongst the highest recipients of antibiotics, understanding the drivers of parental decisions towards their children's antibiotic use is imperative for the development of strategies to assist parents in making more informed decisions. This is particularly relevant to the decisions of parents living in resource-limited settings. This study explored the perspectives and practices of parents living in a rural setting about the use of antibiotics in their children., Methods: Three focus group interviews were conducted with 10 parents of children under 12yrs, living in rural and remote areas of the Northern Territory, Australia. A focus group guide was developed to facilitate discussions, which was informed by the Theory of Planned Behaviour. Transcripts were analysed abductively using thematic analysis., Findings: Four themes emerged explaining factors that contributed to parent decisions about their children's antibiotic use: 1) Parental knowledge, attitudes and decision making; 2) Perceptions of antimicrobial resistance; 3) Healthcare challenges; 4) Behaviours with antibiotics. While parents demonstrated accurate knowledge of the indications for antibiotic use, their decisions about the need for antibiotics were often driven by fear of serious illness. This fear was exacerbated by resource limitations associated with living in a resource-limited setting. Additional drivers of parental decision making included what parents have read or heard from both medical and non-medical networks, underscoring the importance of Social Norms in predicting behavioural intentions., Conclusion: Mothers living in remote areas experiencing reduced access to health services may make decisions about antibiotic use out of fear and based on the advice of their personal network when they perceive their child is vulnerable to a health threat. Findings from this study provide guidance for future research in the prediction of antibiotic use behaviours and for context-specific interventions., Competing Interests: NO authors have competing interests., (Copyright: © 2023 Marsh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Drug self-administration and intracranial self-stimulation (ICSS) are two preclinical behavioral procedures used to predict abuse potential of drugs, and abuse-related drug effects in both procedures are thought to depend on increased mesolimbic dopamine (DA) signaling. Drug self-administration and ICSS yield concordant metrics of abuse potential across a diverse range of drug mechanisms of action. The "rate of onset," defined as the velocity with which a drug produces its effect once administered, has also been implicated as a determinant of abuse-related drug effects in self-administration procedures, but this variable has not been systematically examined in ICSS. Accordingly, this study compared ICSS effects produced in rats by three DA transporter inhibitors that have different rates of onset (fastest to slowest: cocaine, WIN-35428, RTI-31) and that produced progressively weaker metrics of abuse potential in a drug self-administration procedure in rhesus monkeys. Additionally, in vivo photometry using the fluorescent DA sensor dLight1.1 targeted to the nucleus accumbens (NAc) was used to assess the time course of extracellular DA levels as a neurochemical correlate of behavioral effects. All three compounds produced ICSS facilitation and increased DA levels assessed by dLight. In both procedures, the rank order of onset rate was cocaine > WIN-35428 > RTI-31; however, in contrast to monkey drug self-administration results, maximum effects did not differ across compounds. These results provide additional evidence that drug-induced increases in DA drive ICSS facilitation in rats and illustrate the utility of both ICSS and photometry to evaluate the time course and magnitude of abuse-related drug effects in rats., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rationale: Tianeptine is a mu-opioid receptor (MOR) agonist with increasing reports of abuse in human populations. Preclinical data regarding the abuse potential and other opioid-like adverse effects of tianeptine at supratherapeutic doses are sparse., Objectives: The present study evaluated tianeptine in a rat model of abuse potential assessment and in mouse models of motor, gastrointestinal, and respiratory adverse effects., Methods: Abuse potential was assessed in adult male Sprague-Dawley rats using an intracranial self-stimulation (ICSS) procedure to determine effects of acute and repeated tianeptine on responding for electrical brain stimulation. Male ICR mice were used to determine the effects of tianeptine in assays of locomotor behavior and gastrointestinal motility. Male Swiss-Webster mice were monitored for respiratory changes using whole-body plethysmography., Results: In rats, acute tianeptine produced weak and delayed evidence for abuse-related ICSS facilitation at an intermediate dose (10 mg/kg, IP) and pronounced, naltrexone-preventable ICSS depression at a higher dose (32 mg/kg, IP). Repeated 7-day tianeptine (10 and 32 mg/kg/day, IP) produced no increase in abuse-related ICSS facilitation, only modest tolerance to ICSS depression, and no evidence of physical dependence. In mice, tianeptine produced dose-dependent, naltrexone-preventable locomotor activation. Tianeptine (100 mg/kg, SC) also significantly inhibited gastrointestinal motility and produced naloxone-reversible respiratory depression., Conclusions: Tianeptine presents as a MOR agonist with resistance to tolerance and dependence in our ICSS assay in rats, and it has lower abuse potential by this metric than many commonly abused opioids. Nonetheless, tianeptine produces MOR agonist-like acute adverse effects that include motor impairment, constipation, and respiratory depression., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Clinical studies suggest that psychedelics exert robust therapeutic benefits in a number of psychiatric conditions including substance use disorder. Preclinical studies focused on safety and efficacy of these compounds are necessary to determine the full range of psychedelics' effects., Objectives: The present study explores the behavioral pharmacology of structurally distinct psychedelics in paradigms associated with serotonin 2A receptor (5-HT 2A R) activation and behavioral disruption in two rodent models. Utilizing the selective 5-HT 2A R antagonist volinanserin, we aimed to provide further pharmacological assessment of psychedelic effects in rodents., Methods: We compared volinanserin (0.0001-0.1 mg/kg) antagonism of the phenethylamine 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 1.0 mg/kg) and the ergoline lysergic acid diethylamide (LSD, 0.32 mg/kg) in preclinical assays predictive of hallucinations (head-twitch response or HTR in mice) and behavioral disruption (intracranial self-stimulation or ICSS in rats). Volinanserin antagonism of the phenethylamine mescaline, the tryptamine psilocybin, and the k-opioid receptor agonist salvinorin A was also evaluated in the rat ICSS assay., Results: Volinanserin had similar potency, effectiveness, and time-course to attenuate DOI-induced HTR in mice and ICSS depression in rats. Volinanserin completely blocked LSD-induced HTR in mice, but not LSD-induced ICSS depression in rats. Volinanserin also reversed ICSS depression by mescaline, but it was only partially effective to reduce the effects of psilocybin, and it exacerbated ICSS depression by salvinorin A., Conclusion: Although hallucination-related HTR behavior induced by phenethylamine, ergoline, and tryptamine psychedelics appears to be 5-HT 2A R-mediated, the receptor(s) responsible for behavioral disruptive effects may differ among these three structural classes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rationale: Clinically relevant pain is often associated with functional impairment and behavioral depression, including depression of social behavior. Moreover, recovery of function is a major goal in pain treatment. We used a recently developed model of operant responding for social interaction in rats to evaluate the vulnerability of social behavior to an experimental pain manipulation and the sensitivity of pain-depressed social behavior to treatment with clinically effective analgesics., Methods: Sprague-Dawley male and female rats were trained to lever press for social access to another rat, and responding was evaluated after treatment with (a) intraperitoneal injection of dilute lactic acid (IP acid; 0.18-5.6%) administered alone as a visceral noxious stimulus, (b) the mu-opioid receptor (MOR) agonist morphine (0.32-10 mg/kg) or nonsteroidal anti-inflammatory drug (NSAID) ketoprofen (10 mg/kg) administered alone, or (c) morphine or ketoprofen administered before IP acid. For comparison, the same treatments were evaluated in separate rats trained to lever press for food delivery., Results: Both IP acid alone and morphine alone more potently decreased responding maintained by social interaction than by food, whereas ketoprofen did not affect responding for either reinforcer. In general, analgesics were most effective to rescue operant responding when relatively low IP acid concentrations produced significant but submaximal behavioral depression; however, morphine was not effective to rescue responding for social interaction., Conclusions: Operant responding maintained by social interaction was more sensitive to pain-related disruption and less responsive to opioid analgesic rescue than food-maintained operant responding. Social behavior may be especially vulnerable to depression by pain states., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Opioid withdrawal is a key driver of opioid addiction and an obstacle to recovery. However, withdrawal effects on opioid reinforcement and mesolimbic neuroadaptation are understudied and the role of sex is largely unknown., Methods: Male (n=13) and female (n=12) rats responded under a fentanyl-vs.-food "choice" procedure during daily 2h sessions. In addition to the daily choice sessions, rats were provided extended access to fentanyl during 12h self-administration sessions. After two weeks of this self-administration regimen, the nucleus accumbens (NAc) and ventral tegmental area (VTA) of a subset of rats were subjected to RNA sequencing. In the remaining rats, a third week of this self-administration regimen was conducted, during which methadone effects on fentanyl-vs.-food choice were determined., Results: Prior to opioid dependence, male and female rats similarly allocated responding between fentanyl and food. Abstinence from extended fentanyl access elicited similar increases in somatic withdrawal signs in both sexes. Despite similar withdrawal signs and extended access fentanyl intake, opioid withdrawal was accompanied by a maladaptive increase in fentanyl choice in males, but not females. Behavioral sex differences corresponded with a greater number of differentially expressed genes in the NAc and VTA of opioid-withdrawn females relative to males. Methadone blocked withdrawal-associated increases in fentanyl choice in males, but failed to further decrease fentanyl choice in females., Conclusions: These results provide foundational evidence of sex-specific neuroadaptations to opioid withdrawal, which may be relevant to the female-specific resilience to withdrawal-associated increases in opioid choice and aid in the identification of novel therapeutic targets., Competing Interests: 5. Financial Disclosures The authors report no biomedical financial interests or potential conflicts of interest.

Myocardial infarction leads to a rapid innate immune response that is ultimately required for repair of damaged heart tissue. We therefore examined circulating monocyte dynamics immediately after reperfusion of the culprit coronary vessel in STEMI patients to determine whether this correlated with level of cardiac injury. A mouse model of cardiac ischemia/reperfusion injury was subsequently used to establish the degree of monocyte margination to the coronary vasculature that could potentially contribute to the drop in circulating monocytes. We retrospectively analyzed blood samples from 51 STEMI patients to assess the number of non-classical (NC), classical, and intermediate monocytes immediately following primary percutaneous coronary intervention. Classical and intermediate monocytes showed minimal change. On the other hand, circulating numbers of NC monocytes fell by approximately 50% at 90 minutes post-reperfusion. This rapid decrease in NC monocytes was greatest in patients with the largest infarct size (P < .05) and correlated inversely with left ventricular function (r = 0.41, P = .04). The early fall in NC monocytes post-reperfusion was confirmed in a second prospective study of 13 STEMI patients. Furthermore, in a mouse cardiac ischemia model, there was significant monocyte adhesion to coronary vessel endothelium at 2 hours post-reperfusion pointing to a specific and rapid vessel margination response to cardiac injury. In conclusion, rapid depletion of NC monocytes from the circulation in STEMI patients following coronary artery reperfusion correlates with the level of acute cardiac injury and involves rapid margination to the coronary vasculature., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Clinically relevant chronic pain is often associated with functional impairment and behavioral depression as an "affective/motivational" sign of pain; however preclinical animal models of inflammatory and neuropathic pain often produce weak evidence of impaired function. We hypothesized that hindpaw mechanical stimulation produced by a requirement to rear on a textured "NOX" plate would punish operant responding in rats treated with intraplantar complete Freund's adjuvant (CFA, a model of inflammatory pain) or the chemotherapeutic paclitaxel (PTX, a model of neuropathic pain) and produce sustained pain-related depression of operant behavior. Male Sprague-Dawley rats were trained under a progressive-ratio (PR) schedule of food-maintained operant responding, then treated with CFA (100 µL in left hindpaw), PTX (2.0 mg/kg IP on alternate days for four total injections; 6.6 mg/kg IV on alternate days for three total injections), or saline vehicle. PR break points and mechanical thresholds for paw withdrawal from von Frey filaments were then tracked for 28 days. Subsequently, rats were tested with the opioid receptor antagonist naltrexone to assess latent sensitization and with the kappa opioid receptor (KOR) agonist U69593 to assess KOR function. CFA produced significant mechanical hypersensitivity for 3 weeks but decreased PR breakpoints for only 1 day. Both IP and IV PTX produced mechanical hypersensitivity for at least three weeks; however, only IV PTX decreased PR breakpoints, and this decrease was not alleviated by morphine. After recovery, naltrexone reinstated mechanical hypersensitivity in CFA- but not PTX-treated rats, and it did not reinstate depression of breakpoints in any group. U69593 dose-dependently decreased PR breakpoints in all groups with no difference between control vs. CFA/PTX groups. These results suggest that rearing on a textured NOX plate was not sufficient to punish operant responding in CFA- and PTX-treated rats despite the presence of sustained mechanical hypersensitivity. The rapid recovery of operant responding could not be attributed to latent sensitization, KOR downregulation, or behavioral tolerance. These results extend the range of conditions under which putative chronic pain manipulations produce weak evidence for depression of operant responding as a sign of the "affective/motivational" component of pain in rats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Negus, Marsh and Townsend.)

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch ( e.g. , human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans ., (Copyright © 2020 Dirksen et al.)

Background: Host-microbe associations underlie many key processes of host development, immunity, and life history. Yet, none of the current research on the central model species Caenorhabditis elegans considers the worm's natural microbiome. Instead, almost all laboratories exclusively use the canonical strain N2 and derived mutants, maintained through routine bleach sterilization in monoxenic cultures with an E. coli strain as food. Here, we characterize for the first time the native microbiome of C. elegans and assess its influence on nematode life history characteristics., Results: Nematodes sampled directly from their native habitats carry a species-rich bacterial community, dominated by Proteobacteria such as Enterobacteriaceae and members of the genera Pseudomonas, Stenotrophomonas, Ochrobactrum, and Sphingomonas. The C. elegans microbiome is distinct from that of the worm's natural environment and the congeneric species C. remanei. Exposure to a derived experimental microbiome revealed that bacterial composition is influenced by host developmental stage and genotype. These experiments also showed that the microbes enhance host fitness under standard and also stressful conditions (e.g., high temperature and either low or high osmolarity). Taking advantage of the nematode's transparency, we further demonstrate that several Proteobacteria are able to enter the C. elegans gut and that an Ochrobactrum isolate even seems to be able to persist in the intestines under stressful conditions. Moreover, three Pseudomonas isolates produce an anti-fungal effect in vitro which we show can contribute to the worm's defense against fungal pathogens in vivo., Conclusion: This first systematic analysis of the nematode's native microbiome reveals a species-rich bacterial community to be associated with C. elegans, which is likely of central importance for our understanding of the worm's biology. The information acquired and the microbial isolates now available for experimental work establishes C. elegans as a tractable model for the in-depth dissection of host-microbiome interactions.

The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca(2+) signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework., (© 2014 Society for Laboratory Automation and Screening.)

Aerobic exercise training (AET) attenuates or reverses pathological cardiac remodeling after insults such as chronic hypertension and myocardial infarction. The phenotype of the pathologically hypertrophied heart depends on the insult; therefore, it is likely that distinct types of pathological hypertrophy require different exercise regimens. However, the mechanisms by which AET improves the structure and function of the pathologically hypertrophied heart are not well understood, and exercise research uses highly inconsistent exercise regimens in diverse patient populations. There is a clear need for systematic research to identify precise exercise prescriptions for different conditions of pathological hypertrophy. Therefore, this review synthesizes existing evidence for the distinct mechanisms by which AET benefits the heart in different pathological hypertrophy conditions, suggests strategic exercise prescriptions for these conditions, and highlights areas for future research., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A growing body of evidence demonstrates the involvement of protein modification with O-linked β-N-acetylglucosamine (O-GlcNAc) in the stress response and its beneficial effects on cell survival. Here we investigated protein O-GlcNAcylation in skeletal muscle cells exposed to oxidative stress and the crosstalk with endogenous antioxidant system. The study focused on antioxidant enzymes superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase 1 (GPX1), and transcriptional regulators proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and forkhead box protein O1 (FOXO1), which play important roles in oxidative stress response and are known to be O-GlcNAc-modified. C2C12 myoblasts were subjected to 24 h incubation with different reagents, including hydrogen peroxide, diethyl maleate, high glucose, and glucosamine, and the inhibitors of O-GlcNAc cycling enzymes. Surprisingly, O-GlcNAc levels were significantly increased only with glucosamine, whilst other treatments showed no effect. Significant changes at the mRNA level were observed with concomitant upregulation of the genes for O-GlcNAc enzymes and stress-related proteins with oxidizing agents and downregulation of these genes with agents promoting O-GlcNAcylation. Our findings suggest a role of O-GlcNAc in the stress response and indicate an inhibitory mechanism controlling O-GlcNAc levels in the muscle cells. This could represent an important homeostatic regulation of the cellular defense system.

Post-translational modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) profoundly affects protein structure, function, and metabolism. Although many skeletal muscle proteins are O-GlcNAcylated, the modification has not been extensively studied in this tissue, especially in the context of exercise. This study investigated the effects of glutathione depletion and acute exercise on O-GlcNAc protein modification in rat skeletal muscle. Diethyl maleate (DEM) was used to deplete intracellular glutathione and rats were subjected to a treadmill run. White gastrocnemius and soleus muscles were analyzed for glutathione status, O-GlcNAc and O-GlcNAc transferase (OGT) protein levels, and mRNA expression of OGT, O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase. DEM and exercise both reduced intracellular glutathione and increased O-GlcNAc. DEM upregulated OGT protein expression. The effects of the interventions were significant 4 h after exercise (P < 0.05). The changes in the mRNA levels of O-GlcNAc enzymes were different in the two muscles, potentially resulting from different rates of oxidative stress and metabolic demands between the muscle types. These findings indicate that oxidative environment promotes O-GlcNAcylation in skeletal muscle and suggest an interrelationship between cellular redox state and O-GlcNAc protein modification. This could represent one mechanism underlying cellular adaptation to oxidative stress and health benefits of exercise.

We investigated the relationship between markers of mitochondrial biogenesis, cell signaling, and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats + DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise; and (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P < 0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P < 0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P < 0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) mRNA compared to the control animals that were exercised (P < 0.05). This study provides novel evidence that by lowering the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis., (© 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Our understanding of the role of protein O-GlcNAcylation in the regulation of the cardiovascular system has increased rapidly in recent years. Studies have linked increased O-GlcNAc levels to glucose toxicity and diabetic complications; conversely, acute activation of O-GlcNAcylation has been shown to be cardioprotective. However, it is also increasingly evident that O-GlcNAc turnover plays a central role in the delicate regulation of the cardiovascular system. Therefore, the goals of this minireview are to summarize our current understanding of how changes in O-GlcNAcylation influence cardiovascular pathophysiology and to highlight the evidence that O-GlcNAc cycling is critical for normal function of the cardiovascular system., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Heart failure treatment currently centers on symptom management, primarily through reductions in systemic blood pressure and fluid retention. The O-linked attachment of β-N-acetylglucosamine to cardiac proteins is increased in cardiovascular disease and heart failure, and O-GlcNAc transferase (OGT) is the enzyme that catalyzes this addition. Deletion of OGT is embryonically lethal, and cardiomyocyte-specific OGT knockdown causes the exacerbation of heart failure. Stem cell therapy is currently a major focus of heart failure research, and it was recently discovered that OGT is intricately involved with stem cell differentiation. This article focuses on the relationship of OGT with epigenetics and pluripotency, and integrates OGT with several emerging areas of heart failure research, including calcium signaling.

Introduction: Dystrophinopathy in the young mdx mouse model of Duchenne muscular dystrophy is comparatively mild, requires induction, and is rarely assessed with tests of systemic muscle function in whole animals., Methods: A modified TREAT-NMD induction protocol was used to evaluate respiratory and exercise performance, starting and ending with maximum oxygen consumption (VO2max ) tests., Results: The initial and/or final VO2max , time to exhaustion, speed at exhaustion, and total expended calories were significantly lower in mdx mice. Episodic VO2 and VCO2 fluctuations occurred during training and resulted in dissociated patterns of VO2 and respiratory exchange ratio (RER). These fluctuations further resulted in significantly greater VO2 coefficient of variation and RER values and lower minimal VO2 values., Conclusions: Quantifying respiratory performance during exercise is a potentially useful means for studying pathophysiology in mdx mice, as it assesses intact animals over time, is more sensitive than some histological markers, and assesses systemic muscle function., (© 2014 Wiley Periodicals, Inc.)

High-fat, low-carbohydrate diets (HFLCD) are often eaten by humans for a variety of reasons, but the effects of such diets on the heart are incompletely understood. We evaluated the impact of HFLCD on myocardial ischemia/reperfusion (I/R) using an in vivo model of left anterior descending coronary artery ligation. Sprague-Dawley rats (300 g) were fed HFLCD (60% calories fat, 30% protein, 10% carbohydrate) or control (CONT; 16% fat, 19% protein, 65% carbohydrate) diet for 2 wk and then underwent open chest I/R. At baseline (preischemia), diet did not affect left ventricular (LV) systolic and diastolic function. Oil red O staining revealed presence of lipid in the heart with HFLCD but not in CONT. Following I/R, recovery of LV function was decreased in HFLCD. HFLCD hearts exhibited decreased ATP synthase and increased uncoupling protein-3 gene and protein expression. HFLCD downregulated mitochondrial fusion proteins and upregulated fission proteins and store-operated Ca(2+) channel proteins. HFLCD led to increased death during I/R; 6 of 22 CONT rats and 16 of 26 HFLCD rats died due to ventricular arrhythmias and hemodynamic shock. In surviving rats, HFLCD led to larger infarct size. We concluded that in vivo HFLCD does not affect nonischemic LV function but leads to greater myocardial injury during I/R, with increased risk of death by pump failure and ventricular arrhythmias, which might be associated with altered cardiac energetics, mitochondrial fission/fusion dynamics, and store-operated Ca(2+) channel expression., (Copyright © 2014 the American Physiological Society.)

Diets high in sugar and saturated fat (Western diet) contribute to obesity and pathophysiology of metabolic syndrome. A common physiological response to obesity is hypertension, which induces cardiac remodeling and hypertrophy. Hypertrophy is regulated at the level of chromatin by repressor element 1-silencing transcription factor (REST), and pathological hypertrophy is associated with reexpression of a fetal cardiac gene program. Reactivation of fetal genes is commonly observed in hypertension-induced hypertrophy; however, this response is blunted in diabetic hearts, partially due to upregulation of the posttranslational modification O-linked-β-N-acetylglucosamine (O-GlcNAc) to proteins by O-GlcNAc transferase (OGT). OGT and O-GlcNAc are found in chromatin-modifying complexes, but it is unknown whether they play a role in Western diet-induced hypertrophic remodeling. Therefore, we investigated the interactions between O-GlcNAc, OGT, and the fetal gene-regulating transcription factor complex REST/mammalian switch-independent 3A/histone deacetylase (HDAC). Five-week-old male C57BL/6 mice were fed a Western (n = 12) or control diet (n = 12) for 2 wk to examine the early hypertrophic response. Western diet-fed mice exhibited fasting hyperglycemia and increased body weight (P < 0.05). As expected for this short duration of feeding, cardiac hypertrophy was not yet evident. We found that REST is O-GlcNAcylated and physically interacts with OGT in mouse hearts. Western blot analysis showed that HDAC protein levels were not different between groups; however, relative to controls, Western diet hearts showed increased REST and decreased ANP and skeletal α-actin. Transcript levels of HDAC2 and cardiac α-actin were decreased in Western diet hearts. These data suggest that REST coordinates regulation of diet-induced hypertrophy at the level of chromatin.

Pathological cardiac hypertrophy activates a suite of genes called the fetal gene program (FGP). Pathological hypertrophy occurs in diabetic cardiomyopathy (DCM); therefore, the FGP is widely used as a biomarker of DCM in animal studies. However, it is unknown whether the FGP is a consistent marker of hypertrophy in rodent models of diabetes. Therefore, we analyzed this relationship in 94 systematically selected studies. Results showed that diabetes induced with cytotoxic glucose analogs such as streptozotocin was associated with decreased cardiac weight, but genetic or diet-induced models of diabetes were significantly more likely to show cardiac hypertrophy (P<0.05). Animal strain, sex, age, and duration of diabetes did not moderate this effect. There were no correlations between the heart weight:body weight index and mRNA or protein levels of the fetal genes α-myosin heavy chain (α-MHC) or β-MHC, sarco/endoplasmic reticulum Ca2+-ATPase, atrial natriuretic peptide (ANP), or brain natriuretic peptide. The only correlates of non-indexed heart weight were the protein levels of α-MHC (Spearman's ρ = 1, P<0.05) and ANP (ρ = -0.73, P<0.05). These results indicate that most commonly measured genes in the FGP are confounded by diabetogenic methods, and are not associated with cardiac hypertrophy in rodent models of diabetes.

Background: The molecular mechanisms of exercise-induced cardioprotection are poorly understood. We recently reported that exercise training down-regulated gene expression of the Ras homolog gene family member A (RhoA). RhoA and its first effectors, the Rho-kinases (ROCK), have already been implicated in the pathogenesis of cardiovascular disease. The aim of this study was to compare the effects of a RhoA/ROCK inhibitor (fasudil) and exercise in the Apolipoprotein E knockout (ApoE(-/-)) mouse model of atherosclerosis., Methods: Four groups of 14 week old ApoE(-/-) mice were randomised as follows (n=12/group): i) sedentary controls (Cont); ii) fasudil (Fas) treatment (100mg/kg bodyweight/day) for 8 weeks; iii) exercise intervention (Ex:free access to running wheel for 8 weeks) and iv) exercise intervention and fasudil treatment (ExFas) for 8 weeks., Results: Phosphorylation of myosin light chain was significantly reduced in the brachiocephalic artery of all treatment groups compared with sedentary controls, implying an inhibitory effect of exercise and fasudil on the RhoA/ROCK pathway. Furthermore, atherosclerotic lesions were significantly smaller in all treatment and intervention groups compared with the control group (Fas: 34.7%, Ex: 48.3%, ExFas: 40.9% less than Control). The intima:media ratio was reduced by both exercise intervention and fasudil treatment alone or in combination (Fas: 23.6%, Ex: 35.5%, ExFas: 43.9% less than Control). Exercise alone and fasudil treatment alone also showed similar effects on plaque composition, increasing both smooth muscle cell and macrophage density., Conclusion: These results suggest that the protective effects of exercise on atherogenesis are similar to the inhibitory effects on the RhoA/ROCK signalling pathway., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Exercise causes physiological cardiac hypertrophy and benefits the diabetic heart. Mammalian switch-independent 3A (mSin3A) and histone deacetylases (HDACs) 1 and 2 regulate hypertrophic genes through associations with the DNA binding proteins repressor element-1 silencing transcription factor (REST) and O-linked β-N-acetylglucosamine transferase (OGT). O-linked β-N-acetylglucosamine (O-GlcNAc) is a glucose derivative that is chronically elevated in diabetic hearts, and a previous study showed that exercise reduces cardiac O-GlcNAc. We hypothesized that O-GlcNAc and OGT would physically associate with mSin3A/HDAC1/2 in the heart, and that this interaction would be altered by diabetes and exercise., Methods: 8-week-old type 2 diabetic db/db (db) and non-diabetic C57 mice were randomized to treadmill exercise or sedentary groups for 1 or 4 weeks., Results: O-GlcNAc was significantly higher in db hearts and increased with exercise. Db hearts showed lower levels of mSin3A, HDAC1, and HDAC2 protein, but higher levels of HDAC2 mRNA and HDAC1/2 deacetylase activity. Elevated HDAC activity was associated with significantly blunted expression of α-actin and brain natriuretic peptide in db hearts. In sedentary db hearts, co-immunoprecipitation assays showed that mSin3A and OGT were less associated with HDAC1 and HDAC2, respectively, compared to sedentary C57 controls; however, exercise removed these differences., Conclusions: These data indicate that diabetes and exercise oppositely affect interactions between pro-hypertrophic transcription factors, and suggest that an increase in total cardiac O-GlcNAc is a mechanism by which exercise benefits type 2 diabetic hearts.

Cardiac hypertrophy induced by pathological stimuli is regulated by a complex formed by the repressor element 1-silencing transcription factor (REST) and its corepressor mSin3A. We previously reported that hypertrophic signaling is blunted by O-linked attachment of β-N-acetylglucosamine (O-GlcNAc) to proteins. Regular exercise induces a physiological hypertrophic phenotype in the heart that is associated with decreased O-GlcNAc levels, but a link between O-GlcNAc, the REST complex, and initiation of exercise-induced cardiac hypertrophy is not known. Therefore, mice underwent a single 15- or 30-min bout of moderate- to high-intensity treadmill running, and hearts were harvested immediately and compared with sedentary controls. Cytosolic O-GlcNAc was lower (P < 0.05) following 15 min exercise with no differences in nuclear levels (P > 0.05). There were no differences in cytosolic or nuclear O-GlcNAc levels in hearts after 30 min exercise (P > 0.05). Cellular compartment levels of O-GlcNAc transferase (OGT, the enzyme that removes the O-GlcNAc moiety from proteins), REST, mSin3A, and histone deacetylases (HDACs) 1, 2, 3, 4, and 5 were not changed with exercise. Immunoprecipitation revealed O-GlcNAcylation of OGT and HDACs 1, 2, 4, and 5 that was not changed with acute exercise; however, exercised hearts did exhibit lower interactions between OGT and REST (P < 0.05) but not between OGT and mSin3A. These data suggest that hypertrophic signaling in the heart may be initiated by as little as 15 min of exercise via intracellular changes in protein O-GlcNAcylation distribution and reduced interactions between OGT and the REST chromatin repressor.

The post-translation attachment of O-linked N-acetylglucosamine, or O-GlcNAc, to serine and threonine residues of nuclear and cytoplasmic proteins is increasingly recognized as a key regulator of diverse cellular processes. O-GlcNAc synthesis is essential for cell survival and it has been shown that acute activation of pathways, which increase cellular O-GlcNAc levels is cytoprotective; however, prolonged increases in O-GlcNAcylation have been implicated in a number of chronic diseases. Glucose metabolism via the hexosamine biosynthesis pathway plays a central role in regulating O-GlcNAc synthesis; consequently, sustained increases in O-GlcNAc levels have been implicated in glucose toxicity and insulin resistance. Studies on the role of O-GlcNAc in regulating cardiomyocyte function have grown rapidly over the past decade and there is growing evidence that increased O-GlcNAc levels contribute to the adverse effects of diabetes on the heart, including impaired contractility, calcium handling, and abnormal stress responses. Recent evidence also suggests that O-GlcNAc plays a role in epigenetic control of gene transcription. The goal of this review is to provide an overview of our current knowledge about the regulation of protein O-GlcNAcylation and to explore in more detail O-GlcNAc-mediated responses in the diabetic heart., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: Increased O-linked attachment of β-N-acetylglucosamine (O-GlcNAc) to proteins has been implicated in the adverse effects of diabetes on the heart, although this has typically been based on models of severe hyperglycemia. Diabetes has also been associated with dysregulation of autophagy, a critical cell survival process; however, little is known regarding autophagy in the diabetic heart or whether this is influenced by O-GlcNAcylation or hemodynamic stress., Main Methods: Young male rats were assigned to control (12% kcal fat/19% protein/69% carbohydrate), high fat diet (60/19/21%) and type 2 diabetic (high fat diet+low dose streptozotocin) groups for 8 weeks, followed by sham or pressure overload surgeries; animals were sacrificed 8 weeks after surgery., Key Findings: A modest increase in arterial pressure resulted in no significant effects on cardiac function in control or high fat groups, while diabetic hearts exhibited contractile dysfunction and increased apoptosis and scar formation. Immunoprecipitation studies revealed, for the first time, that Beclin-1, which plays a critical early role in autophagy, and the anti-apoptotic Bcl-2, are targets for O-GlcNAcylation. Interestingly, we also found that cardiomyocytes isolated from type 2 diabetic db/db mice exhibited a blunted autophagic response and this was at least partially reversed by inhibiting glucose entry into the hexosamine biosynthesis pathway, which regulates O-GlcNAc synthesis. We also found that acutely augmenting O-GlcNAc levels in non-diabetic cardiomyocytes mimicked the effects of diabetes by blunting autophagic signaling., Significance: These data suggest that O-GlcNAc-mediated inhibition of autophagy may contribute to the abnormal response of diabetic hearts to hemodynamic stress., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Store-operated calcium entry (SOCE) is a major Ca(2+) signaling pathway responsible for regulating numerous transcriptional events. In cardiomyocytes SOCE has been shown to play an important role in regulating hypertrophic signaling pathways, including nuclear translocation of NFAT. Acute activation of pathways leading to O-GlcNAc synthesis have been shown to impair SOCE-mediated transcription and in diabetes, where O-GlcNAc levels are chronically elevated, cardiac hypertrophic signaling is also impaired. Therefore the goal of this study was to determine whether changes in cardiomyocyte O-GlcNAc levels impaired the function of STIM1, a widely recognized mediator of SOCE. We demonstrated that acute activation of SOCE in neonatal cardiomyocytes resulted in STIM1 puncta formation, which was inhibited in a dose-dependent manner by increasing O-GlcNAc synthesis with glucosamine or inhibiting O-GlcNAcase with thiamet-G. Glucosamine and thiamet-G also inhibited SOCE and were associated with increased O-GlcNAc modification of STIM1. These results suggest that activation of cardiomyocyte O-GlcNAcylation attenuates SOCE via STIM1 O-GlcNAcylation and that this may represent a new mechanism by which increased O-GlcNAc levels regulate Ca(2+)-mediated events in cardiomyocytes. Further, since SOCE is a fundamental mechanism underlying Ca(2+) signaling in most cells and tissues, it is possible that STIM1 represents a nexus linking protein O-GlcNAcylation with Ca(2+)-mediated transcription.

Background: The molecular mechanisms of exercise training induced cardiovascular protection are poorly understood. There is growing evidence that reactive oxygen species may be involved in a number of these adaptations and that antioxidants may be used to investigate this effect., Objective: To determine the effects of exercise training and/or antioxidant supplementation on myocardial endothelium and vascular endothelium gene expression., Methods: Male Wistar rats were divided into four groups: i) control; ii) exercise trained (90 min of treadmill running 4d per week, 14 weeks); iii) antioxidant-supplemented (α-tocopherol 1000 IU kg(-1) diet and α-lipoic acid 1.6 g kg(-1) diet, mixed with rat chow) and iv) exercise trained and antioxidant-supplemented., Results: cDNA microarray analysis showed diverse expression changes in both left ventricular and coronary artery endothelial cells. In particular, RT-PCR analysis showed that a gene involved in cardiovascular disease progression, Ras homolog gene family member A, was down-regulated by exercise, and up-regulated by antioxidant supplementation in left ventricular endothelial cells. Furthermore, an important gene involved in inflammation, IL-6, was down-regulated by all treatments., Conclusions: Exercise training and/or antioxidant supplementation affects cardiac endothelial cell gene expression, and their effects on genes such as ras homolog gene family member A and IL-6 provides insight into the molecular mechanisms of their influences on cardiovascular diseases., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Aims: Protein O-GlcNAcylation is both a nutrient sensing and cellular stress response that mediates signal transduction in the heart. Chronically elevated O-GlcNAc has been associated with the development of cardiac dysfunction at both the cellular and organ levels in obesity, insulin resistance and diabetes. Development of these pathologies is often attributed to diets high in saturated fat and sugar (a "Western" diet; WES) but a role for O-GlcNAc in diet-induced cardiac dysfunction has not been established. The aims of this study were to examine the effect of chronic consumption of WES on cardiac O-GlcNAcylation and investigate associations of O-GlcNAc with cardiac function and markers of cellular stress., Main Methods: Young male rats received either a control diet (CON; n=9) or WES (n=8) diet for 52 weeks., Key Findings: There was no evidence of cardiac dysfunction, advanced glycation endproduct (AGE) accumulation, pathological cardiac hypertrophy, calcium handling impairment, fibrosis or endoplasmic reticulum stress in WES hearts. However, cardiac O-GlcNAc protein, particularly in the higher molecular weight range, was significantly higher in WES hearts compared to CON (P<0.05). Protein levels of the enzymes that regulate O-GlcNAc attachment were not different between groups; thus, the increased O-GlcNAcylation in WES hearts appears to be due to increased nutrient availability rather than enzymatic regulation of cellular stress., Significance: These data suggest that diets high in saturated fat and sugar may contribute to the adverse effects of metabolic syndrome and diabetes by an O-GlcNAc-mediated process and that this may occur in the absence of overt cellular stress., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Minimizing damage during reperfusion of the heart following an ischemic event is an important part of the recovery process, as is preventing future recurrences; however, restoring blood perfusion to the heart following ischemia can lead to apoptosis, necrosis, and finally, diminished cardiac function. Exercise reduces risk of heart disease and has been shown to improve the recovery of the heart following ischemia and reperfusion. Brief intermittent ischemic events administered prior to or following a myocardial infarction have also been demonstrated to reduce the infarct size and improve cardiac function, thereby providing cardioprotection. Many signaling transduction pathways are known to regulate cardioprotection, including but not limited to calcium regulation, antioxidant scavenging, and kinase activation. Although posttranslational modifications (PTM) such as phosphorylation, O-GlcNAcylation, methylation, and acetylation are essential regulators of these pathways, their contributions are often overlooked in the literature. This review will examine how PTMS are important regulators of cardioprotection and demonstrate why they should be targeted when developing future therapies for the minimization of damage caused by cardiac ischemia and reperfusion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Exercise increases the production of reactive oxygen species (ROS) in skeletal muscle, and athletes often consume antioxidant supplements in the belief they will attenuate ROS-related muscle damage and fatigue during exercise. However, exercise-induced ROS may regulate beneficial skeletal muscle adaptations, such as increased mitochondrial biogenesis. We therefore investigated the effects of long-term antioxidant supplementation with vitamin E and α-lipoic acid on changes in markers of mitochondrial biogenesis in the skeletal muscle of exercise-trained and sedentary rats., Methods: Male Wistar rats were divided into four groups: 1) sedentary control diet, 2) sedentary antioxidant diet, 3) exercise control diet, and 4) exercise antioxidant diet. Animals ran on a treadmill 4 d · wk at ∼ 70%VO2max for up to 90 min · d for 14 wk., Results: Consistent with the augmentation of skeletal muscle mitochondrial biogenesis and antioxidant defenses, after training there were significant increases in peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) messenger RNA (mRNA) and protein, cytochrome C oxidase subunit IV (COX IV) and cytochrome C protein abundance, citrate synthase activity, Nfe2l2, and SOD2 protein (P < 0.05). Antioxidant supplementation reduced PGC-1α mRNA, PGC-1α and COX IV protein, and citrate synthase enzyme activity (P < 0.05) in both sedentary and exercise-trained rats., Conclusions: Vitamin E and α-lipoic acid supplementation suppresses skeletal muscle mitochondrial biogenesis, regardless of training status.

Evidence for the role of oxidative stress in the pathogenesis of cardiovascular disease (CVD) is primarily based on experimental and observational human studies. The aim of this review is to examine the observational longitudinal studies that have investigated the relationship between oxidative stress biomarkers and CVD. Fifty-one studies were identified with twenty-six of these measuring oxidized (Ox)-LDL, fifteen assessing myeloperoxidase, seven using lipid peroxidation measures and three quantifying protein oxidation. Results of studies using Ox-LDL have been equivocal with sixteen of the twenty-six studies reporting that this measure is predictive of cardiovascular events. These inconsistent results are not explained by differences in the study populations (primary or secondary CVD) or the type of assay used (auto or monoclonal antibodies). Six of the seven lipid peroxidation, and two of three protein oxidation studies found associations. Twelve of fifteen studies assessing the role of myeloperoxidase reported it to be predictive of CVD. However, issues surrounding the specificity of myeloperoxidase as a marker of oxidative stress and the small number of research groups reporting these results, limit this finding. In summary, the ability of oxidative stress biomarkers to predict CVD has yet to be established. Furthermore, it is important to note that the methods used to assess oxidative stress in these studies are indirect, and the evidence that the various methods actually reflect oxidative stress in vivo is limited., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Patients with diabetes have a much greater risk of developing heart failure than non-diabetic patients, particularly in response to an additional hemodynamic stress such as hypertension or infarction. Previous studies have shown that increased glucose metabolism via the hexosamine biosynthesis pathway (HBP) and associated increase in O-linked-β-N-acetylglucosamine (O-GlcNAc) levels on proteins contributed to the adverse effects of diabetes on the heart. Therefore, in this study we tested the hypothesis that diabetes leads to impaired cardiomyocyte hypertrophic and cell signaling pathways due to increased HBP flux and O-GlcNAc modification on proteins. Cardiomyocytes isolated from type 2 diabetic db/db mice and non-diabetic controls were treated with 1 μM ANG angiotensin II (ANG) and 10 μM phenylephrine (PE) for 24 h. Activation of hypertrophic and cell signaling pathways was determined by assessing protein expression levels of atrial natriuretic peptide (ANP), α-sarcomeric actin, p53, Bax and Bcl-2 and phosphorylation of p38, ERK and Akt. ANG II and PE significantly increased levels of ANP and α-actin and phosphorylation of p38 and ERK in the non-diabetic but not in the diabetic group; phosphorylation of Akt was unchanged irrespective of group or treatment. Constitutive Bcl-2 levels were lower in diabetic hearts, while there was no difference in p53 and Bax. Activation of the HBP and increased protein O-GlcNAcylation in non-diabetic cardiomyocytes exhibited a significantly decreased hypertrophic signaling response to ANG or PE compared to control cells. Inhibition of the HBP partially restored the hypertrophic signaling response of diabetic cardiomyocytes. These results suggest that activation of the HBP and protein O-GlcNAcylation modulates hypertrophic and cell signaling pathways in type 2 diabetes.

Acute increases in O-linked β-N-acetylglucosamine (O-GlcNAc) levels of cardiac proteins exert protective effects against ischemia-reperfusion (I/R) injury. One strategy to rapidly increase cellular O-GlcNAc levels is inhibition of O-GlcNAcase (OGA), which catalyzes O-GlcNAc removal. Here we tested the cardioprotective efficacy of two novel and highly selective OGA inhibitors, the NAG-thiazoline derivatives NAG-Bt and NAG-Ae. Isolated perfused rat hearts were subjected to 20 min global ischemia followed by 60 min reperfusion. At the time of reperfusion, hearts were assigned to the following four groups: 1) untreated control; 2) 50 μM NAG-Bt; 3) 100 μM NAG-Bt; or 4) 50 μM NAG-Ae. All treatment groups significantly increased total O-GlcNAc levels (P < 0.05 vs. control), and this was significantly correlated with improved contractile function and reduced cardiac troponin I release (P < 0.05). Immunohistochemistry of normoxic hearts showed intense nuclear O-GlcNAc staining and higher intensity at Z-lines with colocalization of O-GlcNAc and the Z-line proteins desmin and vinculin. After I/R, there was a marked loss of both cytosolic and nuclear O-GlcNAcylation and disruption of normal striated Z-line structures. OGA inhibition largely preserved structural integrity and attenuated the loss of O-GlcNAcylation; however, nuclear O-GlcNAc levels remained low. Immunoblot analysis confirmed ∼50% loss in both nuclear and cytosolic O-GlcNAcylation following I/R, which was significantly attenuated by OGA inhibition (P < 0.05). These data provide further support for the notion that increasing cardiac O-GlcNAc levels by inhibiting OGA may be a clinically relevant approach for ischemic cardioprotection, in part, by preserving the integrity of O-GlcNAc-associated Z-line protein structures.