Acoplamiento excitación-metabolismo : desde la despolarización de la membrana celular a los cambios en la función mitocondrial en músculo esquelético adulto

Tesis para optar al grado de doctor en ciencias biomédicasIntroducción: Es importante dilucidar los mecanismos que vinculan la contracción de la fibra con la síntesis de ATP para comprender la función del músculo esquelético. Las mitocondrias muestran una arquitectura particular en las fibras musculares esqueléticas. Una gran fracción reside entre el sarcolema y las miofibrillas, esta fracción se conoce como mitocondrias subsarcolemales. Una segunda población se encuentra entre las miofibrillas, donde la producción de ATP es esencial para la contracción e interactúa estrechamente con las estructuras de la tríada. Esta segunda población se conoce como mitocondrias intermiofibrilares. Sin embargo, la interacción funcional entre estas poblaciones mitocondriales aún es desconocida. Objetivo: Determinar el efecto de la despolarización de la membrana celular sobre el manejo del Ca2+ mitocondrial y su relación con la función mitocondrial en fibras musculares esqueléticas adultas. Métodos: Se utilizaron fibras musculares aisladas desde el músculo flexor digitorum brevis (FDB). Los niveles de Ca2+ citoplasmáticos y en la matriz mitocondrial se evaluaron utilizando herramientas moleculares específicas para cada compartimento. El papel de los canales intracelulares de Ca2+ se valoró usando tanto inhibidores farmacológicos específicos como herramientas genéticas. El consumo de O2 y el potencial de membrana mitocondrial se detectaron utilizando SeaHorse System y sondas fluorescentes respectivamente. Resultados: En las fibras musculares esqueléticas aisladas, la despolarización aumentó los niveles citoplasmáticos y mitocondriales de Ca2+. La captación mitocondrial de Ca2+ requirió de la activación tanto de los canales de Ca2+ IP3R como RyR1. Además, la inhibición de cualquiera de los dos canales disminuyó la tasa de consumo de O2 basal, pero solo la inhibición de RyR1 previno el aumento del consumo de O2 ligado a la síntesis de ATP. Las señales de Ca2+ inducidas por despolarización se acompañaron de una reducción en el potencial de membrana mitocondrial en las mitocondrias subsarcolemales; Las señales de Ca2+ se propagaron hacia las mitocondrias intermiofibrilares, donde el potencial de membrana mitocondrial aumentó. Los resultados son compatibles con una propagación dependiente de Ca2+ del potencial de membrana mitocondrial, desde la superficie hacia el centro de la fibra muscular. Conclusión: La despolarización de la fibra muscular esquelética aumenta el consumo mitocondrial de O2 y los niveles de Ca2+ mitocondrial, este último efecto depende tanto de la activación del IP3R así como del RyR1. La activación del RyR1, pero no del IP3R, es necesaria para el aumento del consumo de O2 inducido por despolarización. La propagación del potencial de membrana mitocondrial desde la superficie hacia el centro de la fibra podría tener un papel crítico en el control del metabolismo mitocondrial, tanto en reposo como después de la despolarización, formando parte de un proceso llamado acoplamiento "excitación-metabolismo" en las fibras del músculo esquelético. Este mecanismo sería fundamental para mantener la bioenergética del músculo esquelético, manteniendo el equilibrio entre los requerimientos y la síntesis de ATP, permitiendo así sostener la función muscular frente a las demandas ambientales.Introduction: It is important to elucidate the mechanisms that link the contraction of skeletal muscle fiber with the synthesis of ATP to understand the function of skeletal muscle. Mitochondria show a particular architecture in the skeletal muscle fibers. A large fraction resides between the sarcolemma and the myofibrils, this fraction is known as subsarcolemmal mitochondria. A second population is found among the myofibrils, where the production of ATP is essential for contraction and interacts closely with the structures of the triad. This second population is known as intermyofibrillar mitochondria. However, the functional interaction between these mitochondrial populations is still unknown. Objective: To determine the effect of depolarization of the cell membrane on mitochondrial Ca2+ handling and its relationship with mitochondrial function in adult skeletal muscle fibers. Methods: Muscle fibers isolated from flexor digitorum brevis muscle (FDB) were used. Cytoplasmic and Ca2+ levels in the mitochondrial matrix were evaluated using specific molecular tools for each compartment. The role of intracellular Ca2+ channels was assessed using both specific pharmacological inhibitors and genetic tools. O2 consumption and mitochondrial membrane potential were detected using SeaHorse System and fluorescent probes respectively. Results: In isolated skeletal muscle fibers, depolarization increased the cytoplasmic and mitochondrial Ca2+ level. Mitochondrial Ca2+ uptake required the activation of both IP3R and RyR1 Ca2+ channels. In addition, the inhibition of either intracellular Ca2+ channels decreased the basal O2 consumption rate, but only the inhibition of RyR1 prevented the increase of O2 consumption linked to the ATP synthesis. Ca2+ signals induced by depolarization were accompanied by a reduction in mitochondrial membrane potential in subsarcolemmal mitochondria; The Ca2+ signals propagated to the intermyofibrillar mitochondria, where the mitochondrial membrane potential increased. The results are compatible with a Ca2+-dependent propagation of the mitochondrial membrane potential, from the surface to the center of the muscle fiber. Conclusion: The depolarization of the skeletal muscle fiber increases the mitochondrial O2 consumption rate and the levels of mitochondrial Ca2+, this latter effect depends both on the activation of the IP3R as well as the RyR1. The activation of RyR1, but not of IP3R, is necessary for the increase of O2 consumption induced by depolarization. The propagation of the mitochondrial membrane potential from the surface towards the fiber center could have a critical role in the control of mitochondrial metabolism, both at rest and after depolarization, forming part of a process termed "excitation metabolism coupling " in the skeletal muscle fibers. This mechanism would be fundamental to maintain the bioenergetics of the skeletal muscle, maintaining the balance between the requirements and the synthesis of ATP, thus allowing sustaining the muscle function in face of the environmental demands

Skeletal muscle excitation-metabolism coupling

Mitochondria represent the main source of ATP in skeletal muscle and mitochondria activity increases after muscle fiber depolarization. The regulation of mitochondrial function during contraction in skeletal muscle, however, is poorly understood. Skeletal muscle has a particular distribution of mitochondria where three distinct populations can be recognized. The most studied populations are the ones positioned deep into the myofibers between the myofibrils (intermyofibrillar mitochondria), and that located immediately beneath sarcolemma (subsarcolemmal mitochondria); a less studied population locates covering the myonuclei, as a continuation of the subsarcolemmal population. All mitochondria populations undergo fusion and fission events and intermyofibrillar mitochondria are interconnected; mitochondrial communication is necessary to maintain not only the energetic homeostasis of the muscle but its contractile function, as well. The mechanism supporting communication between subsarcolemmal and intermyofibrillar mitochondria is unknown. The recently described MCU complex of proteins has provided a new insight into the role of calcium as a regulator of mitochondrial function. Whether the different mitochondria populations have different calcium handling capacity and whether mitochondria Ca 2+ has a role in energy transmission along the mitochondria network are intriguing issues that emerge when studying the link between electrical stimulation of the muscle fiber and the mitochondria metabolic output

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-p47phox levels were significantly upregulated by exercise in flexor digitorum brevis (FDB). Moreover, exercise significantly increased NOX2 complex assembly (p47phox-gp91phox 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-6) 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

ROS Production via P2Y(1)-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

Artículo de publicación ISIDuring exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7-dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y(1) receptor agonist, induced a large signal while UTPyS (P2Y(2) agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y(1). Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y(1) receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.CONICYT-PIA ACT111 FONDECYT 111046

Is mitochondrial dysfunction a common root of noncommunicable chronic diseases?

Mitochondrial damage is implicated as a major contributing factor for a number of noncommunicable chronic diseases such as cardiovascular diseases, cancer, obesity, and insulin resistance/type 2 diabetes. Here, we discuss the role of mitochondria in maintaining cellular and whole-organism homeostasis, the mechanisms that promote mitochondrial dysfunction, and the role of this phenomenon in noncommunicable chronic diseases. We also review the state of the art regarding the preclinical evidence associated with the regulation of mitochondrial function and the development of current mitochondria-targeted therapeutics to treat noncommunicable chronic diseases. Finally, we give an integrated vision of how mitochondrial damage is implicated in these metabolic diseases.Agencia Nacional de Investigación y Desarrollo (ANID), Chile FONDECYT 1161156 FONDECYT 1200490 FONDAP 15130011 Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) American Heart Association 19TPA34920001 United States Department of Health & Human Services National Institutes of Health (NIH) - USA RO1HD101006 National Health and Medical Research Council of Australia APP1142403 VID, Universidad de Chile ENL 18/19 Australian Research Council DP18010348

Working model.

<p>Electrical stimulation in adult FDB fibers activates Cav1.1 with each depolarizing event. This activation in turn induces ATP release via PnX1 channel. These events will trigger in turn a signaling cascade where, through activation of P2Y₁ receptors, PI3K and PLC and consequent PKC activation induces NOX2 activation and ROS production. Dotted lines show signaling pathways already described. Solid lines show our observations.</p

Electrical stimulation and exogenous ATP induced increase of ROS production.

<p>Muscle fibers were isolated, load with DCF (30min) and stimulated with electrical stimulation (ES) (20Hz) or exogenous ATP (10μM). A. Representative image in pseudo-color of a cell loaded with DCF in control condition (upper panel) and stimulated with ES (lower panel). B, representative traces of DCF fluorescence under control or stimulated with ES or exogenous ATP. C, muscle cells were stimulated with ES or ATP and the slope of fluorescence is shows (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>). D, muscle cells were stimulated with ES at 20Hz or 90Hz and the slope of fluorescence is show (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">Materials and Methods</a></i>) (n = 4, *p<0.05, **p<0.01).</p

Electrical stimulation induced ROS production via ATP release and purinergic receptor.

<p>Muscle fibers were isolated, load with DCF fluorescence (30 min) under control or stimulated, in the absence or presence of different inhibitors (30 min of incubation). A, representative traces of DCF fluorescence under control or stimulated, in absence or presence of CBX (10μM). B, slope of fluorescence from muscle cells stimulated with ES (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>), in the absence or presence of CBX. C, representative traces of DCF fluorescence under control or stimulated, in the absence or presence of suramin (10μM). D, slope of fluorescence from muscle cells stimulated with ES, in the absence or presence of suramin. (n = 6), **p<0.01, ***p<0.001.</p

Exogenous ATP increases H₂O₂ production via P2Y₁ purinergic receptor.

<p>Muscle fibers were isolated and transfected with HyPer plasmid, 24h post transfection the cells were stimulated. A, H₂O₂ generation was measured before and after ATP (10μM) addition. Left panel shows a representative image of Hyper transfection, right panel image fluorescence in pseudo-color. The scale bar represents 50μm. B, kinetics of extracellular ATP-induced H₂O_2. C, muscle fibers were transfected with with HyPer plasmid and stimulated with ES in absence or presence of apyrase (2U/ml). Maximal fluorescence was plotted D, C. Effect of Mrs2365, Mrs2179, UTPγS or exogenous ATP, maximal fluorescence was plotted (n = 4), *p<0.05, **p<0.01.</p

Electrical stimulation and exogenous ATP increase ROS production via NOX2.

<p>Muscle fibers were isolated, loaded with DCF (30min) and stimulated with electrical stimulation (ES) or exogenous ATP A, representative traces of DCF fluorescence under control conditions or stimulated with ES in the absence or presence of apocynin. B, Muscle cells were stimulated with ES and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">Materials and Methods</a></i>). C, Representative traces of DCF fluorescence under control conditions or stimulated with ES in the absence or presence of gp91ds-TAT or scrambled peptide. D, muscle cells were stimulated with ES and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>). E, representative traces of DCF fluorescence under control or stimulated with exogenous ATP in the absence or presence of gp91ds-TAT or scrambled peptide. F, muscle cells were stimulated with ES and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>)(n = 5, *p<0.05, **p<0.01, ***p<0.001).</p