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Chapter 5

Importantly TNFa also stimulates IL-6 expression. IL-6 is a potent inducer of the acute phase response and is produced by T cells, macrophages, fibroblasts, endothelial and muscle cells. Homodimerization of gp130 triggers the Jak/ STAT signaling cascade and the SHP2/Erk Map kinase cascade to regulate proinflammatory gene expression (including NFkB). IL-6 also induces a decrease in the phosphorylation of ribosomal S6 kinase (p70S6K1), suggesting that cytokines can also negatively regulate translational e ciency.

5.2.2.5Mechanisms of Action: Neurotrophins

Neurotrophins are a family of growth factors first identified for the ability to induce neurogrowth in a trophic fashion.17–19 They have recently emerged as regulators of muscle physiology. Nerve Growth Factor (NGF), Cilliary Neurotrophic Factor (CNF) and Neuregulin have all been shown to be produced in muscle and to act in an autocrine fashion. As a class they appear to promote synthesis while inhibiting degradation; however, the specific pathways remain to be established. Nonetheless, all bind to a receptor at the plasma membrane, which results in receptor dimerization (e.g. similar to myostatin). One member of the receptor complex is a protein tyrosine kinase family and is therefore able to signal via the same PI3K/Akt pathway used by insulin. These receptors also appear able to signal via protein kinase C (PKC), via activation of phospholipase C (PLC), which presumably also activates Ca21 signaling within the muscle.

5.2.2.6Mechanisms of Action: Acetylcholine (ACh)/Ca21

Neuronal input in the form of ACh is required for proper development and maintenance of muscle.19–21 Indeed, denervation promotes rapid atrophy of skeletal muscles showing the critical importance of the nerve-muscle axis in the maintenance of muscle mass.

ACh acts to promote synthesis and inhibit degradation, although the precise mechanisms by which this is achieved are obscure. Presumably as ACh induces plasma membrane depolarization and Ca21 induced Ca21 release from the sarcoplasmic reticulum to regulate contraction, this same mechanism regulates proteostasis. Intramuscular calcium can regulate calcineurin, which has been shown to regulate the activity of NFAT. NFAT, when dephosphorylated by calcineurin, can bind other transcription factors, such as myogenic enhancer factor 2 (MEF2) and thereby promote synthesis of specific proteins. Importantly, over-expression of activated calcineurin has been shown to activate genes associated with type I, slow fibers, thus suggesting that muscle activity can induce specific fiber type transformation toward slow fiber formation. Consistent with this possibly being associated with neuronal input, endurance exercise (e.g. sustained muscle activity) also results in transformation toward a slow phenotype while denervation and disuse results in slow to fast fiber transformation. This suggests not only that ACh regulates specific muscle protein synthesis but also that ACh inputs must be maintained for the synthesis to be sustained. Intramuscular Ca21 also can activate CaMK and PKC.

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Both of these kinases have been shown to phosphorylate histone deacetylase (HDAC) proteins, which relieve HDAC inhibition of MEF and thus provide a second, complementary mechanism by which Ca21 downstream of ACh can regulate specific muscle gene expression.

However, the role of Ca21 is complex. Ca21 concentrations are actually elevated in many situations of muscle-wasting including sepsis and denervation and, perhaps paradoxically, under these conditions, Ca21-dependent protein kinases act to suppress protein synthesis via eukaryotic elongation factor 2 (eEF2K) mediated mechanisms to suppress peptide elongation. Moreover there are Ca21-responsive proteases such as the calpains, which may explain increases in protein breakdown. Thus it may be speculated that the amplitude and frequency of Ca21 currents are important in the outcome of proteostasis.

5.2.2.7Mechanisms of Action: Extracellular Matrix

Physical attachment to the extracellular matrix is important to prevent muscle cell apoptosis and to allow for proper functioning of the muscle.22,23 Several attachment complexes exist and thus several mechanisms for regulation of muscle proteostasis likely exist. However, to date the only evidence for modulation of muscle proteostasis in humans is via integrin-containing attachment complexes. These complexes contain an alpha and beta integrin and together they bind to components of the extracellular matrix. There are roughly 150 proteins thought to associate with these complexes in man and several cause limb girdle muscular dystrophy when mutant (myotilin LGMD1A, caveolin LGMD1C, calpain-3 LGMD2A, telethonin LGMD2G and titin LGMD2J). Importantly another of these proteins, focal adhesion kinase (FAK), has been shown to increase in amount and activity when muscle hypertrophies and decrease in amount and activity when muscle atrophies (both in response to use). Additional details of how FAK modulates proteostasis remain to be elucidated although interaction of the cell surface integrin receptors with extracellular matrix proteins results in the activation of intracellular signaling pathways, including p70S6K1, which positively a ects mRNA translation. In addition it has been suggested that integrin complexes cluster insulin and other growth factor receptors together, presumably in an appropriate microdomain of the plasma membrane to allow them to be in the right location to receive signal from outside of the cell. Lastly, integrin complexes are well known in other cells to control the synthesis of the extracellular matrix thereby creating both the signal that activates them and a feedback mechanism, perhaps akin to the autocrine growth factors. It seems likely that other attachment complexes will emerge as key regulators of proteostasis.

5.2.2.8Mechanisms of Action: Amino Acids (AA)

Dietary protein contains AA, which are key extracellular signals in the regulation of muscle proteostasis.24 Importantly, AA act not only as substrates or