Electrical Pulse Stimulation Protects C2C12 Myotubes against Hydrogen Peroxide-Induced Cytotoxicity via Nrf2/Antioxidant Pathway.

Skeletal muscle contraction evokes numerous biochemical alterations that underpin exercise benefits. This present study aimed to elucidate the mechanism for electrical pulse stimulation (EPS)-induced antioxidant adaptation in C2C12 myotubes. We found that EPS significantly upregulated Nrf2 and a broad array of downstream antioxidant enzymes involved in multiple antioxidant systems. These effects were completely abolished by pretreatment with a ROS scavenger, N-acetylcysteine. MitoSOX-Red, CM-H2DCFDA, and EPR spectroscopy revealed a significantly higher ROS level in mitochondria and cytosol in EPS cells compared to non-stimulated cells. Seahorse and Oroboros revealed that EPS significantly increased the maximal mitochondrial oxygen consumption rate, along with an upregulated protein expression of mitochondrial complexes I/V, mitofusin-1, and mitochondrial fission factor. A post-stimulation time-course experiment demonstrated that upregulated NQO1 and GSTA2 last at least 24 h following the cessation of EPS, whereas elevated ROS declines immediately. These findings suggest an antioxidant preconditioning effect in the EPS cells. A cell viability study suggested that the EPS cells displayed 11- and 36-fold higher survival rates compared to the control cells in response to 2 and 4 mM H2O2 treatment, respectively. In summary, we found that EPS upregulated a large group of antioxidant enzymes in C2C12 myotubes via a contraction-mitochondrial-ROS-Nrf2 pathway. This antioxidant adaptation protects cells against oxidative stress-associated cytotoxicity.

Effects of Acute and Chronic Gabapentin Treatment on Cardiovascular Function of Rats.

Gabapentin (GBP), a GABA analogue, is primarily used as an anticonvulsant for the treatment of partial seizures and neuropathic pain. Whereas a majority of the side effects are associated with the nervous system, emerging evidence suggests there is a high risk of heart diseases in patients taking GBP. In the present study, we first used a preclinical model of rats to investigate, firstly, the acute cardiovascular responses to GBP (bolus i.v. injection, 50 mg/kg) and secondly the effects of chronic GBP treatment (i.p. 100 mg/kg/day × 7 days) on cardiovascular function and the myocardial proteome. Under isoflurane anesthesia, rat blood pressure (BP), heart rate (HR), and left ventricular (LV) hemodynamics were measured using Millar pressure transducers. The LV myocardium and brain cortex were analyzed by proteomics, bioinformatics, and western blot to explore the molecular mechanisms underlying GBP-induced cardiac dysfunction. In the first experiment, we found that i.v. GBP significantly decreased BP, HR, maximal LV pressure, and maximal and minimal dP/dt, whereas it increased IRP-AdP/dt, Tau, systolic, diastolic, and cycle durations (* p < 0.05 and ** p < 0.01 vs. baseline; n = 4). In the second experiment, we found that chronic GBP treatment resulted in hypotension, bradycardia, and LV systolic dysfunction, with no change in plasma norepinephrine. In the myocardium, we identified 109 differentially expressed proteins involved in calcium pathways, cholesterol metabolism, and galactose metabolism. Notably, we found that calmodulin, a key protein of intracellular calcium signaling, was significantly upregulated by GBP in the heart but not in the brain. In summary, we found that acute and chronic GBP treatments suppressed cardiovascular function in rats, which is attributed to abnormal calcium signaling in cardiomyocytes. These data reveal a novel side effect of GBP independent of the nervous system, providing important translational evidence to suggest that GBP can evoke adverse cardiovascular events by depression of myocardial function.

MRP8/14 Is a Molecular Signature Triggered by Dopamine in HIV Latent Myeloid Targets That Increases HIV Transcription and Distinguishes HIV+ Methamphetamine Users with Detectable CSF Viral Load and Brain Pathology.

There is a significant overlap between HIV infection and substance-use disorders. Dopamine (DA) is the most abundantly upregulated neurotransmitter in methamphetamine abuse, with receptors (DRD1-5) that are expressed by neurons as well as by a large diversity of cell types, including innate immune cells that are the targets of HIV infection, making them responsive to the hyperdopaminergic environment that is characteristic of stimulant drugs. Therefore, the presence of high levels of dopamine may affect the pathogenesis of HIV, particularly in the brain. The stimulation of HIV latently infected U1 promonocytes with DA significantly increased viral p24 levels in the supernatant at 24 h, suggesting effects on activation and replication. Using selective agonists to different DRDs, we found that DRD1 played a major role in activating viral transcription, followed by DRD4, which increased p24 with a slower kinetic rate compared to DRD1. Transcriptome and systems biology analyses led to the identification of a cluster of genes responsive to DA, where S100A8 and S100A9 were most significantly correlated with the early increase in p24 levels following DA stimulation. Conversely, DA increased the expression of these genes' transcripts at the protein level, MRP8 and MRP14, respectively, which form a complex also known as calprotectin. Interestingly, MRP8/14 was able to stimulate HIV transcription in latent U1 cells, and this occurred via binding of the complex to the receptor for an advanced glycosylation end-product (RAGE). Using selective agonists, both DRD1 and DRD4 increased MRP8/14 on the surface, in the cytoplasm, as well as secreted in the supernatants. On the other hand, while DRD1/5 did not affect the expression of RAGE, DRD4 stimulation caused its downregulation, offering a mechanism for the delayed effect via DRD4 on the p24 increase. To cross-validate MRP8/14 as a DA signature with a biomarker value, we tested its expression in HIV+ Meth users' postmortem brain specimens and peripheral cells. MRP8/14+ cells were more frequently identified in mesolimbic areas such as the basal ganglia of HIV+ Meth+ cases compared to HIV+ non-Meth users or to controls. Likewise, MRP8/14+ CD11b+ monocytes were more frequent in HIV+ Meth users, particularly in specimens from participants with a detectable viral load in the CSF. Overall, our results suggest that the MRP8 and MRP14 complex may serve as a signature to distinguish subjects using addictive substances in the context of HIV, and that this may play a role in aggravating HIV pathology by promoting viral replication in people with HIV who use Meth.

Quantitative Proteomics Identifies Novel Nrf2-Mediated Adaptative Signaling Pathways in Skeletal Muscle Following Exercise Training.

Exercise training (ExT) improves skeletal muscle health via multiple adaptative pathways. Nrf2 is a principal antioxidant transcription factor responsible for maintaining intracellular redox homeostasis. In this study, we hypothesized that Nrf2 is essential for adaptative responses to ExT and thus beneficial for muscle. Experiments were carried out on male wild type (WT) and iMS-Nrf2flox/flox inducible muscle-specific Nrf2 (KO) mice, which were randomly assigned to serve as sedentary controls (Sed) or underwent 3 weeks of treadmill ExT thus generating four groups: WT-Sed, WT-ExT, KO-Sed, and KO-ExT groups. Mice were examined for exercise performance and in situ tibialis anterior (TA) contractility, followed by mass spectrometry-based proteomics and bioinformatics to identify differentially expressed proteins and signaling pathways. We found that maximal running distance was significantly longer in the WT-ExT group compared to the WT-Sed group, whereas this capacity was impaired in KO-ExT mice. Force generation and fatigue tolerance of the TA were enhanced in WT-ExT, but reduced in KO-ExT, compared to Sed controls. Proteomic analysis further revealed that ExT upregulated 576 proteins in WT but downregulated 207 proteins in KO mice. These proteins represent pathways in redox homeostasis, mitochondrial respiration, and proteomic adaptation of muscle to ExT. In summary, our data suggest a critical role of Nrf2 in the beneficial effects of SkM and adaptation to ExT.