106 L.V. Thompson Using proteomic technology (mass spectrometry and bioinformatics) to identify the proteins susceptible to CML-modification, the CML-modified proteins are critical enzymes involved in energy production (Fig. 14e). Creatine kinase, carbonic anhy- drase III, b-enolase, actin, and voltage-dependent anion channel 1 are susceptible to CML-modification, with b-enolase showing an accumulation of CML with age in skeletal muscle. Because lysines are at the exposed surface of b-enolase, the protein may function as a scavenger of CML, sparing other proteins from AGE- modification and potential functional impairment. b-enolase appears to be a good candidate as a scavenger because glycation of this protein has minimal impairment on cellular physiology (glycolytic flux). The significance of glycation of other skeletal muscle proteins on muscle func- tion is unknown, yet in vitro studies show that glycation decreases myosin and actin interactions (Ramamurthy et al. 2001). The glycation of myosin is detected in the skeletal muscle of aged rats (Syrovy and Hodny 1992). Interestingly, glycation of purified myosin from young rats decreases actin motility and also decreases K + - activated and actin-activated ATPase activities (1). Thus, modification of lysine- rich nucleotide- and actin-binding regions of the myosin molecule is a possible mechanism for the functional loss. In summary, the advancement of experimental technologies, quantitative pro- teomics and bioinformatics, identifies possible underlying mechanism responsible for the aging muscle phenotype. Thus, it will be possible to generate new hypoth- eses on ROS-induced mechanisms of post-translational chemical modifications (e.g., carbonylation) as well as possible connections between protein modifications and cellular functions already known to be impaired in aging muscle. These numer- ous hypotheses provide targets for future testing, a step closer to understanding the role of protein post-translational chemical modification in aging muscle decline. It should be noted that with aging other oxidative modifications might accumulate and/or a site-specific amino acid modification of critical residues on these proteins could adversely affect function and contribute to muscle weakness. Additionally, an important limitation in the characterization of modified proteins from aged tissue is the fact that the data provide only a snapshot of a dynamic process, as proteins are constantly being synthesized and degraded in most tissues. Lastly, current knowledge about post-translational modification, and the techniques available to measure them, may not permit the quantitative analysis of all potential post-translational modifica- tions of a given protein of interest as well as its functional characterization. 8 Age-Related Changes in Protein Expression Levels 8.1 Myosin and Actin Stoichiometry between myosin and actin is critical for skeletal muscle contractility. Maintenance of the stoichiometry between myosin and actin depends on the balance between the protein synthesis and protein degradation. With aging, there is evidence 107Age-Related Decline in Actomyosin Structure and Function for decreased myosin heavy chain synthesis rates and a loss in the regulation of the proteasome, the main protease responsible for degrading myofibrillar proteins. Thus, changes in rates of synthesis or degradation could lead to protein-specific declines in either actin or myosin content. Detailed experiments, in both animal and human, show age-related decreases in myosin but not actin content in muscles composed of MHC type II (D’Antona et al. 2003; Thompson et al. 2006). The reduction in myosin protein expression without a change in actin content alters the optimal stoichiometry, leading to a decrease in the number of active cross-bridges contributing to force generation. In contrast, MHC content was unaffected by age in muscles composed of type I MHC isoform or composed of both type I and type II MHC isoforms indicating muscle-specific molecular changes (Moran et al. 2005; Thompson et al. 2006). Advanced pro- teomic technology has made possible analysis of age-related changes in the whole muscle proteome, yielding differentially expressed proteins with age (up-regulation and down-regulation). The comparison of results for different muscles shows that changes in the expression levels of contractile proteins are muscle specific. The main consequence of changes in expression levels of myosin and other contractile proteins is a change in stoichiometry. Thus, changes in protein stoichiometry may provide a mechanism for the observed aging muscle phenotypes (i.e., weakness in the fast-twitch muscle compared to the slow-twitch muscle). Another mechanism that may explain age-related muscle dysfunction is a shift in skeletal muscle protein isoforms. As noted earlier in this chapter, myosin is a hexamer composed of two heavy chains, two regulatory light chains and two essential light chains such that specific protein isoforms confer contractility (e.g., MHC type II fibers contract faster than MHC type I fibers). Single permeabilized fiber experiments evaluating contractility combined with micro-analysis of isoform composition with SDS-PAGE detect age-related shifts in isoforms that are muscle and fiber-dependent, but these results do not explain the total changes in muscle contractility. 8.2 Muscle Proteome-Protein Expression Over the past 4 years there is evidence of age-related changes in the whole skeletal muscle proteome. In two studies, using mass spectrometry to identify proteins, the analyses of the proteomes detect proteins differently expressed with age (Gelfi et al. 2006; Piec et al. 2005). In both studies, the expression levels for all three myosin light chains were down-regulated. Although more studies are needed to draw conclusions about the changes in the whole skeletal muscle proteome with age, a comparison of results for the two identified studies shows that changes in the expression levels of contractile proteins are muscle specific. As noted earlier, the main consequence of changes in expression levels of contractile proteins is a change in the stoichiometry, which could provide one of the explanations of age-related changes in contractile function. 108 L.V. Thompson 9 Conclusion Reduced muscle function and its attendant decrease in physical performance with age is a significant public health problem. Oxidative damage to key skeletal muscle proteins may be a contributing factor in sarcopenia. Age-related changes in the interaction between the contractile proteins actin and myosin provide some insights about potential molecular mechanisms responsible for age-related alterations in contractility. However, conclusive results require a more complete determination of the extent and location of oxidized sites, with parallel assessment of functional interactions of the proteins. 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Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness, DOI 10.1007/978-90-481-9713-2_6, © Springer Science+Business Media B.V. 2011 Abstract Aging is associated with decreasing strength that can lead to impaired performance of daily living activities in the elderly. Functional and structural decline in the neuromuscular system has been recognized as a cause of this impairment and loss of independence, but the age-related loss of strength is greater than the loss of muscle mass in mammals, including humans, and the underlying mechanisms remain only partially understood. This chapter focuses on skeletal muscle excitation-contraction uncoupling (ECU), external calcium-dependent skeletal muscle contraction, the role of JP-45 and other recently discovered molecules of the muscle T-tubule-sarcoplasmic reticulum junction (triad) in excitation-contraction coupling (ECC), the neural influence of skeletal muscle, and the role of trophic factors–particularly insulin-like growth factor-I (IGF-1)–in structural and functional modifications of the motor unit and the neuromuscular junction with aging. A better understanding of the triad proteins involved in muscle ECC and nerve/muscle interactions and their regulation will lead to more rational interventions to delay or prevent muscle weakness with aging. Keywords Skeletal muscle • Aging • Sarcopenia • Insulin-like growth factor 1 • Denervation O. Delbono (*) Departments of Internal Medicine, Section on Gerontology and Geriatric Medicine, Department of Physiology and Pharmacology, Molecular Medicine and Neuroscience Programs, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA e-mail: odelbono@wfubmc.edu Excitation-Contraction Coupling Regulation in Aging Skeletal Muscle Osvaldo Delbono 114 O. Delbono 1 Age-related Decrease in Strength is Greater than the Decrease in Muscle Mass in Humans Weakness with old age can be partially attributed to a well-recognized decrease in muscle mass. Some studies in humans directly relate this diminished strength to muscle atrophy (Kent-Braun and Ng 1999), while others find that it is greater than the decrease in muscle mass (Lynch et al. 1999). For example, the decline in nor- malized force (force/muscle mass, Nm/kg) in the knee extensors has been found to follow a curve, starting at about 40 years and declining by about 28% from 40–49 to 70–79 years (Lynch et al. 1999). In vitro studies of single human muscle fiber contractility also reveal a decrease in specific force (force/cross-sectional area) with age (Frontera et al. 2000a). Therefore, the intrinsic force-generating capacity of the skeletal muscle per contractile unit may be impaired in aging mammals, including humans. Postulated mechanisms include alterations to the excitation-contraction coupling process (Delbono et al. 1995; Renganathan et al. 1998; Wang et al. 2000) and decreased actin-myosin cross-bridge stability (Lowe et al. 2002). For a review, see (Payne and Delbono 2004). 2 Excitation-Contraction Uncoupling Skeletal muscle contraction is initiated by action potentials generated in the motor neuron and conducted via its axons, culminating in release of acetylcholine at the motor-end plate. Acetylcholine binds to nicotinic acetylcholine receptors, leading to an increase in sodium and potassium conductance in the end-plate membrane. End-plate potentials at the muscle membrane generate action potentials that are conducted to the sarcolemmal infoldings (T-tubules). The transduction of changes in sarcolemmal potential to elevated intracellular calcium concentration is a key event that precedes muscle contraction (Dulhunty 2006). Electro-mechanical transduction in muscle cells requires the participation of the dihydropyridine receptor (DHPR) (Schneider and Chandler 1973) located at the sarcolemmal T-tubule. The DHPR is a voltage-gated L-type Ca 2+ channel (dihydro- pyridine-sensitive), and its activation evokes Ca 2+ release from an intracellular store (SR) through ryanodine-sensitive calcium channels (RyR1) into the myoplasm. The functional consequence of the reduced number, function, or interaction of these receptors is reduced intracellular calcium mobilization and force development (Delbono et al. 1997). Calcium binds to troponin C, leading to cross-linkages between actin and myosin and sliding of thin-on-thick filaments to produce force (Loeser and Delbono 2009). Uncoupling of the excitation-contraction machinery is a major factor in age-dependent decline in the force- generating capacity of indi- vidual cells (Delbono 2002). Aging muscle fibers exhibit less specific force than those from young-adult or middle- aged animals but similar endurance and recovery from fatigue (Gonzalez et al. 2000b) 115Excitation-Contraction Coupling Regulation in Aging Skeletal Muscle (González and Delbono 2001a, b). Whether excitation-contraction uncoupling results from altered neural control of muscle gene expression is not known. However, a series of studies support this concept. First, denervation results in a significant decrease in DHPR functional expression and alterations in excitation- contraction coupling in skeletal muscle from adult rats (Delbono 1992). Second, nerve crush leads to reduced levels of mRNA-encoding DHPR subunits and RyR1 in muscle (Ray et al. 1995), and studies show that both DHPR and RyR1 expression depend on skeletal muscle innervation (Kyselovic et al. 1994; Pereon et al. 1997b). Third, during development, DHPR mRNA levels change in relation to fiber inner- vation (Chaudari and Beam 1993). Fourth, myotube depolarization triggers the appearance of (+)-[ 3 H]PN 200–110 binding sites (Pauwels et al. 1987). Finally, exercise and chronic stimulation in vivo increase DHPR expression in homogenates of soleus and EDL muscles (Saborido et al. 1995; Pereon et al. 1997a). Thus, fiber- type composition, DHPR and RyR1, and excitation-contraction coupling seem to depend on nerve stimulation and muscle activity. We are starting to understand how nerve stimulation of muscle activity influences muscle phenotype and the specific sarcolemmal-nuclear signaling pathways involved in muscle gene expression at different ages. Increasing evidence points to a decline in neural influence on skeletal muscle at later ages (Messi and Delbono 2003), leading to changes in muscle composition that result in excitation-contrac- tion uncoupling (Payne and Delbono 2004). 3 IGF-1 Regulates Skeletal Muscle Excitation-contraction Coupling IGF-1 may affect functional interactions between nerve and muscle by regulating transcription of the DHPRa 1S gene (Zheng et al. 2001). Although the DHPRa1 subunit is critical to excitation-contraction coupling, the basic mechanisms regu- lating its gene expression are unknown. To understand them, we isolated and sequenced the 1.2-kb 5¢ flanking-region fragment immediately upstream of the mouse DHPRa 1S gene (Zheng et al. 2002). Luciferase reporter constructs driven by different promoter regions of that gene were used for transient transfection assays in muscle C2C12 cells. We found that three regions, corresponding to the CREB, GATA-2, and SOX-5 consensus sequences within this flanking region, are important for DHPRa 1S gene transcription, and antisense oligonucleotides against them significantly reduced charge movement in C2C12 cells (Zheng et al. 2002). This study demonstrates that the transcription factors CREB, GATA- 2, and SOX-5 play a significant role in the expression of skeletal muscle DHPRa 1S . Whether IGF-1 regulates these transcription factors and subsequent expression of the DHPRa 1S gene is not known. Using a approach similar to that described above (Zheng et al. 2002), we investigated the effects of IGF-1 on various pro- moter deletion/luciferase reporter constructs. They were transfected into C2C12 . and aging on rat semimembranosus muscle and myosin. Journal of Applied Physiology, 101, 873–880. 113 G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness, DOI 10.1007/978-90-481-9713-2_6,. aging muscle phenotypes (i.e., weakness in the fast-twitch muscle compared to the slow-twitch muscle) . Another mechanism that may explain age-related muscle dysfunction is a shift in skeletal muscle. better understanding of the triad proteins involved in muscle ECC and nerve /muscle interactions and their regulation will lead to more rational interventions to delay or prevent muscle weakness