Fibroblast growth factor 21 is expressed and secreted from skeletal muscle following electrical stimulation via extracellular ATP activation of the PI3K/Akt/mTOR signaling pathway.

Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a "myokine", synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as "Excitation-Transcription coupling". eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF21, through eATP signaling and PI3K/Akt pathway. Our results show that electrical stimulation increases both mRNA and protein (intracellular and secreted) levels of FGF21, dependent on an extracellular ATP signaling mechanism in skeletal muscle. Using pharmacological inhibitors, we demonstrated that FGF21 production and secretion from muscle requires the activation of the P2YR/PI3K/Akt/mTOR signaling pathway. These results confirm skeletal muscle as a source of FGF21 in physiological conditions and unveil a new molecular mechanism for regulating FGF21 production in this tissue. Our results will allow to identify new molecular targets to understand the regulation of FGF21 both in physiological and pathological conditions, such as exercise, aging, insulin resistance, and Duchenne muscular dystrophy, all characterized by an alteration in both FGF21 levels and ATP signaling components. These data reinforce that eATP signaling is a relevant mechanism for myokine expression in skeletal muscle.

Mechanical Disturbance of Osteoclasts Induces ATP Release That Leads to Protein Synthesis in Skeletal Muscle through an Akt-mTOR Signaling Pathway.

Muscle and bone are tightly integrated through mechanical and biochemical signals. Osteoclasts are cells mostly related to pathological bone loss; however, they also start physiological bone remodeling. Therefore, osteoclast signals released during bone remodeling could improve both bone and skeletal muscle mass. Extracellular ATP is an autocrine/paracrine signaling molecule released by bone and muscle cells. Then, in the present work, it was hypothesized that ATP is a paracrine mediator released by osteoclasts and leads to skeletal muscle protein synthesis. RAW264.7-derived osteoclasts were co-cultured in Transwell® chambers with flexor digitorum brevis (FDB) muscle isolated from adult BalbC mice. The osteoclasts at the upper chamber were mechanically stimulated by controlled culture medium perturbation, resulting in a two-fold increase in protein synthesis in FDB muscle at the lower chamber. Osteoclasts released ATP to the extracellular medium in response to mechanical stimulation, proportional to the magnitude of the stimulus and partly dependent on the P2X7 receptor. On the other hand, exogenous ATP promoted Akt phosphorylation (S473) in isolated FDB muscle in a time- and concentration-dependent manner. ATP also induced phosphorylation of proteins downstream Akt: mTOR (S2448), p70S6K (T389) and 4E-BP1 (T37/46). Exogenous ATP increased the protein synthesis rate in FDB muscle 2.2-fold; this effect was blocked by Suramin (general P2X/P2Y antagonist), LY294002 (phosphatidylinositol 3 kinase inhibitor) and Rapamycin (mTOR inhibitor). These blockers, as well as apyrase (ATP metabolizing enzyme), also abolished the induction of FDB protein synthesis evoked by mechanical stimulation of osteoclasts in the co-culture model. Therefore, the present findings suggest that mechanically stimulated osteoclasts release ATP, leading to protein synthesis in isolated FDB muscle, by activating the P2-PI3K-Akt-mTOR pathway. These results open a new area for research and clinical interest in bone-to-muscle crosstalk in adaptive processes related to muscle use/disuse or in musculoskeletal pathologies.

Serious Games as a Method for Enhancing Learning Engagement: Student Perception on Online Higher Education During COVID-19.

The COVID-19 pandemic has enforced social isolation in many countries worldwide, which forced teachers at all levels of education, including the university context, to adapt new teaching strategies. This study presents a method developed in this regard, that is, serious games were used as a complement to synchronous online classes to ensure the continuity of pedagogical activities in a physiology course at Universidad Andrés Bello, Chile. Using serious games is a strategy in the field of gamification, which is a commonly used learning strategy for online teaching as necessitated by COVID-19. This study is quantitative in nature and conducted a questionnaire survey on 108 second-year undergraduate nursing students to determine their perception about this innovation. The results demonstrate that the students well valued the proposed pedagogical innovative model in terms of motivation and engagement. Moreover, they reported that the model can serve as a meaningful learning experience. These perceptions suggest that the model is an efficient strategy for implementing the physiology curricula in the context of online teaching. Moreover, the results imply that the model should be applied to other courses and disciplines in the undergraduate program and provide support that it is a valid strategy for face-to-face teaching. Lastly, the finding points to the potential of the model to be explored as a learning strategy in the age of education post-COVID-19.

Oxoisoaporphines and Aporphines: Versatile Molecules with Anticancer Effects.

Cancer is a disease that involves impaired genome stability with a high mortality index globally. Since its discovery, many have searched for effective treatment, assessing different molecules for their anticancer activity. One of the most studied sources for anticancer therapy is natural compounds and their derivates, like alkaloids, which are organic molecules containing nitrogen atoms in their structure. Among them, oxoisoaporphine and sampangine compounds are receiving increased attention due to their potential anticancer effects. Boldine has also been tested as an anticancer molecule. Boldine is the primary alkaloid extract from boldo, an endemic tree in Chile. These compounds and their derivatives have unique structural properties that potentially have an anticancer mechanism. Different studies showed that this molecule can target cancer cells through several mechanisms, including reactive oxygen species generation, DNA binding, and telomerase enzyme inhibition. In this review, we summarize the state-of-art research related to oxoisoaporphine, sampangine, and boldine, with emphasis on their structural characteristics and the relationship between structure, activity, methods of extraction or synthesis, and anticancer mechanism. With an effective cancer therapy still lacking, these three compounds are good candidates for new anticancer research.

Early molecular response and microanatomical changes in the masseter muscle and mandibular head after botulinum toxin intervention in adult mice.

BACKGROUND: Masseter muscle paralysis induced by botulinum toxin type A (BoNTA) evokes subchondral bone loss in mandibular heads of adult rats and growing mice after 4 weeks. However, the primary cellular and molecular events leading to altered bone remodeling remain unexplored. Thus, the aim of the current work has been to assess the molecular response that precedes the early microanatomical changes in the masseter muscle and subchondral bone of the mandibular head in adult mice after BoNTA intervention. METHODS: A pre-clinical in vivo study was performed by a single intramuscular injection of 0.2 U BoNTA in the right masseter (experimental) of adult BALB/c mice. The contralateral masseter was injected with vehicle (control). Changes in mRNA levels of molecular markers of bone loss or muscle atrophy/regeneration were addressed by qPCR at day 2 or 7, respectively. mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL) was assessed in mandibular heads, whilst mRNA levels of Atrogin-1/MAFbx, MuRF-1 and Myogenin were addressed in masseter muscles. In order to identify the early microanatomical changes at day 14, fiber diameters in transversal sections of masseter muscles were quantified, and histomorphometric analysis was used to determine the bone per tissue area and the trabecular thickness of subchondral bone of the mandibular heads. RESULTS: An increase of up to 4-fold in RANKL mRNA levels were detected in mandibular heads of the BoNTA-injected sides as early as 2 days after intervention. Moreover, a 4-6 fold increase in Atrogin-1/MAFbx and MuRF-1 and an up to 25 fold increase in Myogenin mRNA level were detected in masseter muscles 7 days after BoNTA injections. Masseter muscle mass, as well as individual muscle fiber diameter, were significantly reduced in BoNTA-injected side after 14 days post-intervention. At the same time, in the mandibular heads from the treated side, the subchondral bone loss was evinced by a significant reduction in bone per tissue area (-40%) and trabecular thickness (-55%). CONCLUSIONS: Our results show that masseter muscle paralysis induced by BoNTA leads to significant microanatomical changes by day 14, preceded by molecular changes as early as 2 days in bone, and 7 days in muscle. Therefore, masseter muscle atrophy and subchondral bone loss detected at 14 days are preceded by molecular responses that occur during the first week after BoNTA intervention.

NOX2 Inhibition Impairs Early Muscle Gene Expression Induced by a Single Exercise Bout.

Reactive oxygen species (ROS) participate as signaling molecules in response to exercise in skeletal muscle. However, the source of ROS and the molecular mechanisms involved in these phenomena are still not completely understood. The aim of this work was to study the role of skeletal muscle NADPH oxidase isoform 2 (NOX2) in the molecular response to physical exercise in skeletal muscle. BALB/c mice, pre-treated with a NOX2 inhibitor, apocynin, (3 mg/kg) or vehicle for 3 days, were swim-exercised for 60 min. Phospho-p47(phox) levels were significantly upregulated by exercise in flexor digitorum brevis (FDB). Moreover, exercise significantly increased NOX2 complex assembly (p47(phox)-gp91(phox) interaction) demonstrated by both proximity ligation assay and co-immunoprecipitation. Exercise-induced NOX2 activation was completely inhibited by apocynin treatment. As expected, exercise increased the mRNA levels of manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx), citrate synthase (CS), mitochondrial transcription factor A (tfam) and interleukin-6 (IL-I6) in FDB muscles. Moreover, the apocynin treatment was associated to a reduced activation of p38 MAP kinase, ERK 1/2, and NF-κB signaling pathways after a single bout of exercise. Additionally, the increase in plasma IL-6 elicited by exercise was decreased in apocynin-treated mice compared with the exercised vehicle-group (p < 0.001). These results were corroborated using gp91-dstat in an in vitro exercise model. In conclusion, NOX2 inhibition by both apocynin and gp91dstat, alters the intracellular signaling to exercise and electrical stimuli in skeletal muscle, suggesting that NOX2 plays a critical role in molecular response to an acute exercise.

Characterization of a multiprotein complex involved in excitation-transcription coupling of skeletal muscle.

BACKGROUND: Electrical activity regulates the expression of skeletal muscle genes by a process known as "excitation-transcription" (E-T) coupling. We have demonstrated that release of adenosine 5'-triphosphate (ATP) during depolarization activates membrane P2X/P2Y receptors, being the fundamental mediators between electrical stimulation, slow intracellular calcium transients, and gene expression. We propose that this signaling pathway would require the proper coordination between the voltage sensor (dihydropyridine receptor, DHPR), pannexin 1 channels (Panx1, ATP release conduit), nucleotide receptors, and other signaling molecules. The goal of this study was to assess protein-protein interactions within the E-T machinery and to look for novel constituents in order to characterize the signaling complex. METHODS: Newborn derived myotubes, adult fibers, or triad fractions from rat or mouse skeletal muscles were used. Co-immunoprecipitation, 2D blue native SDS/PAGE, confocal microscopy z-axis reconstruction, and proximity ligation assays were combined to assess the physical proximity of the putative complex interactors. An L6 cell line overexpressing Panx1 (L6-Panx1) was developed to study the influence of some of the complex interactors in modulation of gene expression. RESULTS: Panx1, DHPR, P2Y2 receptor (P2Y2R), and dystrophin co-immunoprecipitated in the different preparations assessed. 2D blue native SDS/PAGE showed that DHPR, Panx1, P2Y2R and caveolin-3 (Cav3) belong to the same multiprotein complex. We observed co-localization and protein-protein proximity between DHPR, Panx1, P2Y2R, and Cav3 in adult fibers and in the L6-Panx1 cell line. We found a very restricted location of Panx1 and Cav3 in a putative T-tubule zone near the sarcolemma, while DHPR was highly expressed all along the transverse (T)-tubule. By Panx1 overexpression, extracellular ATP levels were increased both at rest and after electrical stimulation. Basal mRNA levels of the early gene cfos and the oxidative metabolism markers citrate synthase and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were significantly increased by Panx1 overexpression. Interleukin 6 expression evoked by 20-Hz electrical stimulation (270 pulses, 0.3 ms each) was also significantly upregulated in L6-Panx1 cells. CONCLUSIONS: We propose the existence of a relevant multiprotein complex that coordinates events involved in E-T coupling. Unveiling the molecular actors involved in the regulation of gene expression will contribute to the understanding and treatment of skeletal muscle disorders due to wrong-expressed proteins, as well as to improve skeletal muscle performance.

Insulin elicits a ROS-activated and an IP3-dependent Ca²âº release, which both impinge on GLUT4 translocation.

Insulin signaling includes generation of low levels of H2O2; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H2O2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H2O2 increased the translocation of GLUT4 with an exofacial Myc-epitope tag between the first and second transmembrane domains (GLUT4myc), an effect additive to that of insulin. The anti-oxidants N-acetyl L-cysteine and Trolox, the p47(phox)-NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47(phox) knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-ds-tat suppressed insulin-dependent H2O2 production. A ryanodine receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca(2+) release by promoting RyR1 S-glutathionylation. This pathway acts in parallel to insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP3)-activated Ca(2+) channels, in response to activation of phosphatidylinositol 3-kinase and its downstream target phospholipase C, resulting in Ca(2+) transfer to the mitochondria. An inhibitor of IP3 receptors, Xestospongin B, reduced both insulin-dependent IP3 production and GLUT4myc translocation. We propose that, in addition to the canonical α,ß phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca(2+) release and IP3-receptor-mediated mitochondrial Ca(2+) uptake, and that these signals jointly stimulate glucose uptake.