Abstract

Excitation-metabolism (E-M) coupling is the ensemble of mechanisms by which electrical stimulation of skeletal muscle transduce into metabolic changes in the muscle fiber. These mechanisms are idly known and in most part assumed but have not been tested. The knowledge of these mechanisms will have a profound effect on treatment of metabolic diseases and will shed a new light on the effect of exercise on a variety of health problems. Our aim is to understand the cellular basis of the link between electrical stimulation of the muscle cell (one of the constant features of exercise) and the short and long term metabolic adaptations of skeletal muscle as well as the alterations of this process that occur upon metabolic challenges in obesity and aging. Excitation-metabolism coupling will be studied at three different levels: The first level is the effect of electrical stimulation on glucose transport. The general mechanism we have envisaged includes a role of Cav1.1 channels as voltage sensors, activation of pannexin1 channels, ATP release to the extracellular medium, activation of P2Y purinergic receptors, G-protein mediated activation of PI3 kinase and phospholipase C (PLC). Glucose transport could be increased following translocation of GLUT4 to the T- tubule membrane via two different events; one is Akt phosphorylation after PI3 kinase activation and the second one involves PKC activation (possibly by diacylglycerol produced by PLC), phosphorylation of subunits of NADPH oxidase (NOX2), H2O2 production, ryanodine receptor (RyR1) oxidation and local calcium release, needed for GLUT4 translocation. This mechanism needs to be thoroughly tested and molecular tools will be designed to alter local signals. The second level is the “metabolic boost” in the mitochondria after electrical stimulation. This effect follows the same general mechanism outlined in Goal 1; activation of PLC will produce inositol tris phosphate (IP3) which will diffuse to receptors (IP3Rs) located in a particular region of the sarcoplasmic reticulum (SR) in close contact with mitochondria. The localized calcium rise near the opening of the mitochondria calcium uniporter (MCU) leads to mitochondrial calcium increase. Mitochondria calcium levels regulate several enzymes from the Krebs cycle, inducing an increased activity of oxidative phosphorylation which yields to increases in oxygen consumption and ATP production. Furthermore, a retrograde signal appears to originate in mitochondria to increase GLUT4 translocation to the T-tubule membrane and glucose transport. The third level of coupling is the expression of metabolic genes. The level of expression of a number of genes was shown to be regulated by electrical stimulation in a frequency-dependent manner. Among these genes, we have preliminary evidence pointing to extracellular ATP-dependent activation of PI3 kinase as a pivotal node leading to IP3-dependent and IP3-independent gene expression. Proteins involved in oxidative metabolism as citrate synthase are stimulated by low frequency stimulation, while glycolitic enzymes as enolase are repressed. A number of genes will be analyzed and we will differentiate the effect of nuclear calcium signals and other localized calcium signals from a direct effect of extracellular ATP on signaling pathways leading to activation of calcium-independent transcription factors (in particular NFkB). The ensemble of results will allow understanding the long term regulation of metabolism associated to muscle fiber plasticity. Once the excitation-metabolism coupling mechanisms are established in normal rodent muscle, we will explore these mechanisms in models for human conditions in which metabolic responses are altered. One of these models is mice fed a high fat diet. These mice are obese and insulin resistant, mimicking several aspects of the metabolic syndrome in humans. Another model is aging mice, in which loss of muscle mass (sarcopenia) develops and also present insulin resistance. In both models, all tree levels of excitation-metabolism coupling will be assessed and pharmacological actions tending to revert some of the alterations found will be tested.