Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P13 doc

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. An important limitation in the characterization of dam-
aged proteins from muscle 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. Furthermore, current knowledge about post-translational modification
due to oxidative stress, and the techniques available to measure them, may not
permit the quantitative analysis of all potential modifications of a given protein of
interest, as well as its functional characterization. It is likely that the future will see
a significant increase in the number of specific modifications of proteins known,
and an increase in our ability to associate them with specific aging phenotypes.
References
Balaban, R. S., Nemoto, S., Finkel, T. (2005). Mitochondria, oxidants, and aging. Cell, 120, 483–495.
Balagopal, P., Rooyackers, O. E., Adey, D. B., Ades, P. A., Nair, K. S. (1997). Effects of aging on
in vivo synthesis of skeletal muscle myosin heavy-chain and sarcoplasmic protein in humans.
American Journal of Physiology. Endocrinology and Metabolism, 273, E790–E800.
Barany, M. (1967). ATPase activity of myosin correlated with speed of muscle shortening. The
Journal of General Physiology, 50(Suppl), 197–218.
Baumgartner, R. N., Koehler, K. M., Gallagher, D., Romero, L., Heymsfield, S. B., Ross, R. R.,
Garry, P.J., Lindeman R. D. (1998). Epidemiology of sarcopenia among the elderly in New
Mexico. American Journal of Epidemiology, 147, 755–763.
Bobkov, A. A., Bobkova, E. A., Homsher, E., Reisler, E. (1997). Activation of regulated actin by
SH1-modified myosin subfragment 1. Biochemistry, 36, 7733–7738.
Bobkova, E. A., Bobkov, A. A., Levitsky, D. I., Reisler, E. (1999). Effects of SH1 and SH2 modi-
fications on myosin similarities and differences. Biophysical Journal, 76, 1001–1007.
Cadenas, E. (2004). Nitric oxide, mitochondrial function, and cell signaling. Free Radical Biology
and Medicine, 36, S18–S18.
Callahan, L. A., She, Z. W., Nosek, T. M. (2001). Superoxide, hydroxyl radical, and hydrogen
peroxide effects on single-diaphragm fiber contractile apparatus. Journal of Applied Physiology,

90, 45–54.
Cassina, A. M., Hodara, R., Souza, J. M., Thomson, L., Castro, L., Ischiropoulos, H., Freeman B.A.,
Radi, R. (2000). Cytochrome c nitration by peroxynitrite. Journal of Biological Chemistry, 275,
21409–21415.
Crowder, M. S. & Cooke, R. (1984). The effect of myosin sulfhydryl modification on the mechanics
of fiber contraction. Journal of Muscle Research and Cell Motility, 5, 131–146.
D’Antona, G., Pellegrino, M. A., Adami, R., Rossi, R., Carlizzi, C. N., Canepari, M., Saltin, B.,
Bottinelli, R. (2003). The effect of ageing and immobilization on structure and function of
human skeletal muscle fibres. Journal of Physiology-London, 552, 499–511.
109Age-Related Decline in Actomyosin Structure and Function
Dantzig, J. A., Goldman, Y. E., Millar, N. C., Lacktis, J., Homsher, E. (1992). Reversal of the
cross-bridge force-generating transition by photogeneration of phosphate in rabbit psoas
muscle-fibers. Journal of Physiology-London, 451, 247–278.
Dyer, D. G., Dunn, J. A., Thorpe, S. R., Bailie, K. E., Lyons, T. J., McCance, D. R., Baynes, J.W.
(1993). Accumulation of Maillard reaction-products in skin collagen in diabetes and aging.
The Journal of Clinical Investigation, 91, 2463–2469.
Feng, J., Xie, H. W., Meany, D. L., Thompson, L. V., Arriaga, E. A., Griffin, T. J. (2008).
Quantitative proteomic profiling of muscle type-dependent and age-dependent protein carbo-
nylation in rat skeletal muscle mitochondria. The Journals of Gerontology. Series A: Biological
Sciences and Medical Sciences, 63, 1137–1152.
Ferrington, D. A., Husom, A. D., Thompson, L. V. (2005). Altered proteasome structure, function,
and oxidation in aged muscle. Faseb Journal, 19, 644–646.
Frisard, M. & Ravussin, E. (2006). Energy metabolism and oxidative stress – impact on the meta-
bolic syndrome and the aging process. Endocrine, 29, 27–32.
Fugere, N. A., Ferrington, D. A., Thompson, L. V. (2006). Protein nitration with aging in the rat
semimembranosus and soleus muscles. The Journals of Gerontology. Series A: Biological
Sciences and Medical Sciences, 61, 806–812.
Gelfi, C., Vigano, A., Ripamonti, M., Pontoglio, A., Begum, S., Pellegrino, M. A., Grassi, B.,
Bottinelli, R., Wait, R., Cerretelli, P. (2006). The human muscle proteome in aging. Journal of
Proteome Research, 5, 1344–1353.

Hook, P. & Larsson, L. (2000). Actomyosin interactions in a novel single muscle fiber in vitro
motility assay. Journal of Muscle Research and Cell Motility, 21, 357–365.
Hook, P., Sriramoju, V., Larsson, L. (2001). Effects of aging on actin sliding speed on myosin
from single skeletal muscle cells of mice, rats, and humans. American Journal of Physiology.
Cell Physiology, 280, C782–C788.
Husom, A. D., Peters, E. A., Kolling, E. A., Fugere, N. A., Thompson, L. V., Ferrington, D. A.
(2004). Altered proteasome function and subunit composition in aged muscle. Archives of
Biochemistry and Biophysics, 421, 67–76.
Husom, A. D., Ferrington, D. A., Thompson, L. V. (2005). Age-related differences in the adaptive
potential of type I skeletal muscle fibers. Experimental Gerontology, 40, 227–235.
Ingalls, C. P., Warren, G. L., Armstrong, R. B. (1998). Dissociation of force production from
MHC and actin contents in muscles injured by eccentric contractions. Journal of Muscle
Research and Cell Motility, 19, 215–224.
Ji, L. L. (2001). Exercise at old age: does it increase or alleviate oxidative stress? Healthy Aging
for Functional Longevity, 928, 236–247.
Ji, L. L. (2002). Exercise-induced modulation of antioxidant defense. Increasing Healthy Life
Span: Conventional Measures and Slowing the Innate Aging Process, 959, 82–92.
Ji, L. L., Leeuwenburgh, C., Leichtweis, S., Gore, M., Fiebig, R., Hollander, J., Bejma, J. (1998).
Oxidative stress and aging – role of exercise and its influences on antioxidant systems.
Towards Prolongation of the Healthy Life Span, 854, 102–117.
Kaldor, G. & Min, B. K. (1975). Enzymatic studies on skeletal myosin a and actomyosin of aging
rats. Federation Proceedings, 34, 191–194.
Lamb, G. D. & Posterino, G. S. (2003). Effects of oxidation and reduction on contractile function
in skeletal muscle fibres of the rat. Journal of Physiology-London, 546, 149–163.
Li, X. P. & Larsson, L. (1996). Maximum shortening velocity and myosin isoforms in single
muscle fibers from young and old rats. American Journal of Physiology – Cell Physiology,
270, C352–C360.
Lionne, C., Brune, M., Webb, M. R., Travers, F., Barman, T. (1995). Time-resolved measurements
show that phosphate release is the rate-limiting step on myofibrillar ATPases. FEBS Letters,
364, 59–62.

Lionne, C., Iorga, B., Candau, R., Piroddi, N., Webb, M. R., Belus, A., Travers, F., Barman, T.
(2002). Evidence that phosphate release is the rate-limiting step on the overall ATPase of psoas
myofibrils prevented from shortening by chemical cross-linking. Biochemistry, 41,
13297–13308.
110 L.V. Thompson
Liu, Y. B., Fiskum, G., Schubert, D. (2002). Generation of reactive oxygen species by the
mitochondrial electron transport chain. Journal of Neurochemistry, 80, 780–787.
Lowe, D. A., Surek, J. T., Thomas, D. D., Thompson, L. V. (2001). Electron paramagnetic reso-
nance reveals age-related myosin structural changes in rat skeletal muscle fibers. American
Journal of Physiology – Cell Physiology, 280, C540–C547.
Lowe, D. A., Thomas, D. D., Thompson, L. V. (2002). Force generation, but not myosin ATPase
activity, declines with age in rat muscle fibers. American Journal of Physiology – Cell
Physiology, 283, C187–C192.
Lowe, D. A., Husom, A. D., Ferrington, D. A., Thompson, L. V. (2004). Myofibrillar myosin
ATPase activity in hindlimb muscles from young and aged rats. Mechanisms of Ageing and
Development, 125, 619–627.
Marston, S. B. & Taylor, E. W. (1980). Comparison of the myosin and actomyosin ATPase mecha-
nisms of the 4 types of vertebrate muscles. Journal of Molecular Biology, 139, 573–600.
Marx, N., Walcher, D., Ivanova, N., Rautzenberg, K., Jung, A., Friedl, R., Hombach, V.,
de Caterina, R., Basta, G., Wautier, M.P., Wautiers, J.L. (2004). Thiazolidinediones reduces
endothelial expression of receptors for advanced glycation end products. Diabetes, 53,
2662–2668.
McCance, D. R., Dyer, D. G., Dunn, J. A., Bailie, K. E., Thorpe, S. R., Baynes, J. W., Lyons, T.J.
(1993). Maillard reaction-products and their relation to complications in insulin-dependent
diabetes-mellitus. Journal of Clinical Investigation, 91, 2470–2478.
Misselwitz, J., Franke, S., Kauf, E., John, U., Stein, G. (2002). Advanced glycation end products
in children with chronic renal failure and type 1 diabetes. Pediatric Nephrology, 17, 316–321.
Moran, A. L., Warren, G. L., Lowe, D. A. (2005). Soleus and EDL muscle contractility across the
lifespan of female C57BL/6 mice. Experimental Gerontology, 40, 966–975.
Ostdal, H., Skibsted, L. H., Andersen, H. J. (1997). Formation of long-lived protein radicals in the

reaction between H
2
O
2
-activated metmyoglobin and other proteins. Free Radical Biology and
Medicine, 23, 754–761.
Patel, H., Li, X., Karan, H. I. (2003). Amperometric glucose sensors based on ferrocene contain-
ing polymeric electron transfer systems – a preliminary report. Biosensors & Bioelectronics,
18, 1073–1076.
Perkins, W. J., Han, Y. S., Sieck, G. C. (1997). Skeletal muscle force and actomyosin ATPase
activity reduced by nitric oxide donor. Journal of Applied Physiology, 83, 1326–1332.
Piec, I., Listrat, A., Alliot, J., Chambon, C., Taylor, R.G., Bechet, D. (2005) Differential proteome
analysis of aging in rat skeletal muscle. Faseb Journal, 19, 1143–1145.
Prochniewicz, E., Walseth, T. F., Thomas, D. D. (2004). Structural dynamics of actin during active
interaction with myosin: different effects of weakly and strongly bound myosin heads.
Biochemistry, 43, 10642–10652.
Prochniewicz, E., Thomas, D. D., Thompson, L. V. (2005). Age-related decline in actomyosin
function. Journals of Gerontology Series A – Biological Sciences and Medical Sciences, 60,
425–431.
Prochniewicz, E., Thompson, L. V., Thomas, D. D. (2007). Age-related decline in actomyosin
structure and function. Experimental Gerontology, 42, 931–938.
Ramamurthy, B., Hook, P., Jones, A. D., Larsson, L. (2001). Changes in myosin structure and
function in response to glycation. FASEB Journal, 15, 2415–2422.
Reid, M. B. & Durham, W. J. (2002). Generation of reactive oxygen and nitrogen species in con-
tracting skeletal muscle – Potential impact on aging. Increasing Healthy Life Span:
Conventional Measures and Slowing the Innate Aging Process, 959, 108–116.
Roubenoff, R. (2000). Sarcopenia and its implications for the elderly. European Journal of
Clinical Nutrition, 54, S40–S47.
Saxena, A. K., Saxena, P., Wu, X. L., Obrenovich, M., Weiss, M. F., Monnier, V. M. (1999).
Protein aging by carboxymethylation of lysines generates sites for divalent metal and redox

active copper binding: Relevance to diseases of glycoxidative stress. Biochemical and
Biophysical Research Communications, 260, 332–338.
111Age-Related Decline in Actomyosin Structure and Function
Srivastava, S. K. & Kanungo, M. S. (1982). Aging modulates some properties of skeletal myosin
ATPase of rat. Biochemical Medicine, 28, 266–272.
St-Pierre, J., Buckingham, J. A., Roebuck, S. J., Brand, M. D. (2002). Topology of superoxide
production from different sites in the mitochondrial electron transport chain. Journal of
Biological Chemistry, 277, 44784–44790.
Syrovy, I. & Hodny, Z. (1992). Nonenzymatic glycosylation of myosin – effects of diabetes and
aging. General Physiology and Biophysics, 11, 301–307.
Thomas, D. D., Kast, D., Korman, V. L. (2009). Site-directed spectroscopic probes of actomyosin
structural dynamics. Annual Review of Biophysics, 38, 347–369.
Thompson, L. V. (2009). Age-related muscle dysfunction. Experimental Gerontology, 44,
106–111.
Thompson, L. V. & Brown, M. (1999). Age-related changes in contractile properties of single
skeletal fibers from the soleus muscle. Journal of Applied Physiology, 86, 881–886.
Thompson, L. V., Lowe, D. A., Ferrington, D. A., Thomas, D. D. (2001). Electron paramagnetic
resonance: a high-resolution tool for muscle physiology. Exercise and Sport Sciences Reviews,
29, 3–6.
Thompson, L. V., Durand, D., Fugere, N. A., Ferrington, D. A. (2006). Myosin and actin expres-
sion and oxidation in aging muscle. Journal of Applied Physiology, 101, 1581–1587.
Uesugi, N., Sakata, N., Horiuchi, S., Nagai, R., Takeya, M., Meng, J., Saito, T., Takebayashi, S.
(2001). Glycoxidation-modified macrophages and lipid peroxidation products are associated
with the progression of human diabetic nephropathy. American Journal Of Kidney Diseases,
38, 1016–1025.
Verzijl, N., DeGroot, J., Oldehinkel, E., Bank, R. A., Thorpe, S. R., Baynes, J. W., Bayliss, M.T.,
Bijlsma, J.W.J., Lafeber, F., TeKoppele, J.M. (2000). Age-related accumulation of Maillard
reaction products in human articular cartilage collagen. The Biochemical Journal, 350,
381–387.
Viner, R. I., Ferrington, D. A., Williams, T. D., Bigelow, D. J., Schoneich, C. (1999). Protein

modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of
the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle. Biochemical Journal, 340,
657–669.
Viner, R. I., Williams, T. D., Schoneich, C. (1999). Peroxynitrite modification of protein thiols:
Oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in
the sarcoplasmic reticulum Ca-ATPase. Biochemistry, 38, 12408–12415.
Wolkow, C. A. & Iser, W. B. (2006). Uncoupling protein homologs may provide a link between
mitochondria, metabolism and lifespan. Ageing Research Reviews, 5, 196–208.
Yan, L. J. & Sohal, R. S. (1998). Mitochondrial adenine nucleotide translocase is modified oxida-
tively during aging. Proceedings of the National Academy of Sciences of the USA, 95,
12896–12901.
Yates, L. D. & Greaser, M. L. (1983). Quantitative-determination of myosin and actin in rabbit
skeletal-muscle. Journal of Molecular Biology, 168, 123–141.
Zhong, S., Lowe, D. A., Thompson, L. V. (2006). Effects of hindlimb unweighting 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, © 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:
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