CHAPTER 5 The Muscular System: The Control of Muscle Mass 157 The functional role of CaN was fi rst delineated in T-cells, where increased levels of intracellular calcium were found to mediate the interaction between calmodulin and CaN, leading to CaN activation. Mechanistically, cytoplasmic nuclear factor of acti- vated T-cell (NFAT) proteins are dephosphorylated by activated CaN, allowing their translocation into the nucleus, where they act combinatorial with other transcription factors to activate target gene transcription (16,17). Since calcium is known to regulate many cellular processes in striated muscle, the ques- tion arose whether the CaN signaling pathway also functioned in striated muscle. The work of Molkentin and colleagues (18) provided the fi rst evidence that CaN was involved in the devel- opment of cardiac hypertrophy. This was followed by the obser- vation of Chin and associates (19), whose work implicated a role for this enzyme in determining skeletal muscle fi ber type. The latter conclusion was based on the fi nding that treatment of rats with cyclosporin A (CsA), an inhibitor of CaN activity, led to a decrease in the proportion of slow fi bers populating the soleus muscle while the proportion of fast fi bers increased. Addition- ally, cell culture experiments implicated the CaN-NFAT path- way in modulating both skeletal muscle phenotype and the development of IGF-1-induced hypertrophy (16,17).
Since these initial observations, numerous research teams have investigated the role of CaN in determining skeletal muscle fi ber type gene expression and the induc- tion of hypertrophy. This work has led to a proposed model postulating that calcium-mobilizing signals evoked by stimuli—such as chronic low-frequency motor nerve stimulation, MOV, or voluntary wheel running—induce a sustained low-amplitude elevation in intracellular calcium levels and this, in turn, stimulates both the CaN and cal- cium-calmodulin protein kinase (CaMK) signaling path- ways. Activation of these two signaling pathways leads to induction of hypertrophic growth and the transcriptional activation of slow fi ber genes mediated by downstream modulators comprising various members of the NFAT and myocyte enhancer factor-2 (MEF2) transcription fac- tor families (16,17). The transcriptional activity of nuclear MEF2 is enhanced by CaN in two ways: (a) by its dephos- phorylation and (b) by direct interaction with nuclear DNA-bound NFAT. CaMK and polycystic kidney disease 1 (PKD1) are also thought to activate the transcriptional activity of DNA-bound MEF2 by disrupting MEF2’s asso- ciation with class II histone deacetylases (HDACs), which act to suppress the transcriptional activation function of MEF2. Disruption of the class II HDAC-MEF2 interaction results in the ubiquitination and proteasomal degradation of class II HDACs (20,21). However, this overall model is not without controversy.
Although an initial series of studies provided evidence implicating a primary role for CaN in mediating skeletal mus- cle hypertrophy and phenotype, a number of recent studies have shown that although this pathway may play a central role in the development of many forms of cardiac hyper- trophy, it is not necessarily required for the development of skeletal muscle hypertrophy or fi ber-type shifts (17). To more clearly resolve whether CaN plays a role in skeletal muscle hypertrophy and/or phenotypic transitions, gene targeting skeletal muscle to destroy the satellite cell population elimi-
nates MOV-induced hypertrophy and increased numbers of myonuclei (15). On the other hand, although irradiation blocked MOV-induced skeletal muscle hypertrophy, it did not alter the fast-to-slow transition in MyHC gene expres- sion. This raises the question to what extent satellite cells participate in the MOV-induced, fast-to-slow phenotype transition. Although numerous signals have been implicated in satellite cell activation, those directly involved in the MOV response are not well defi ned. Some evidence supports a role for IGF-1 and a macrophage response involving secretion of cytokines (14). Additional work will be required to precisely delineate MOV-induced signals involved in satellite cell acti- vation and to resolve issues regarding cross talk between signaling pathways involved in hypertrophic growth versus phenotype transitions.
TEST YOURSELF
What ability has the adult skeletal muscle lost by being terminally differentiated? How does it make up for this loss? Explain your answer in detail.
Why are satellite cells considered adult stem cells?
What is the location of satellite cells and what signals activate them?
Molecular Pathways for Skeletal
158 SECTION 1 Exercise and Responses of Biologic Systems
3. Increased nuclear degradation of HDACs (21) (a collaborative role for PKD1 with CaMK and CaN) 4. These same perturbations activated a MEF2-
dependent reporter transgene in transgenic mice (termed MEF2-sensor mice [16]) and this effect was blocked by treatment with CsA and by transgenic expression of modulatory calcineurin-interacting protein-1 (a peptide inhibitor of CaN now termed RCAN1) (16) (supports a role for CaN and CaMK) However, conclusions concerning MEF2 involvement in regulating slow fi ber gene expression based on the results obtained with the MEF2-sensor transgenic mice must be viewed with caution because it was recently shown that all four TEAD-1 (also referred to as transcriptional enhancer factor-1 [TEF-1]) family members avidly bind the desmin MEF2 ele- ment used in the MEF2 sensor transgene (see Transcriptional Regulation of the MyHC Gene in Response to MOV [23]). Here again, more work needs to be done to sort out roles for this pathway in the transduction of exercise-induced signals into growth or phenotypic transitions.
mice that lack greater than 80% of their total skeletal muscle CaN were generated (22). These mice were found to display a dramatic impairment in the fast-to-slow fi ber type transition in response to MOV; however, hypertrophic growth was not altered indicating that CaN is not required for MOV-induced skeletal muscle hypertrophy (22).
CALCIUM-CALMODULINPROTEINKINASE (CAMK): A CA -
DEPENDENT SIGNALING PATHWAY The activity of CaMK, like CaN, is regulated by intracellular calcium although the amount and type of calcium signal differs. Whereas CaN is activated by sustained low-amplitude calcium signals, CaMK is presumably activated by short-duration, high-amplitude calcium signals (16,17). As previously described, CaMK is thought to enhance MEF2 transcriptional activity by dis- rupting its interaction with HDACs which function as tran- scriptional repressors. In addition, new evidence has revealed that PKD1 also functions in this capacity (20). The activation of MEF2 has been tied to skeletal muscle fi ber–type gene expression based on a number of observations:
1. MOV, chronic low-frequency nerve stimulation, voluntary wheel running, and overexpression of an activated CaN protein resulted in decreased nuclear MEF2 phosphorylation (16,17) (supports role for CaN)
2. Electrophoretic mobility shift assays, which are designed to examine DNA–protein interaction, have shown that the degree of MEF2 protein binding at MEF2 elements does not change in response to these perturbations (16) (indicates a release of HDAC tran- scriptional repression supporting a role for CaMK)
TEST YOURSELF
You have isolated a new gene that is expressed only in skeletal muscle. Stimulation of intracellular signaling pathway X has been shown to activate a downstream mediator (transcription factor) that confers muscle- specifi c expression to other muscle genes.
Should you assume that the same signaling pathway and transcription factor confers muscle-specifi c expres- sion to your gene?
How might stimulation of a given signaling pathway alter the activity of a DNA-bound transcription factor?
RAS-MITOGEN-ACTIVATEDPROTEINKINASE (MAPK) PATHWAY
The important infl uence of neural input on modulating mus- cle phenotype has been recognized since the early studies of Buller and associates (24), which demonstrated that fast muscles take on slow-twitch contractile properties following cross-innervation with a slow motor neuron and vice versa.
To determine the signaling pathways involved in this process, Murgia and colleagues (25) investigated the role of the mono- meric GTP-binding protein (Ras)-MAPK pathway, previously shown to be activated by electrostimulation of muscle. Use of several Ras mutants allowed these researchers to show that the Ras-MAPK pathway mimics the effects of slow nerve innervation, thereby implicating its role in regulating nerve- dependent slow muscle gene expression (25). A constitutively activated Ras mutant was found to activate the ERK pathway (a MAPK pathway), which reproduced the effect of slow motor nerve input by activating slow myosin (Sm) expression and decreasing fast myosin expression in denervated regeneration muscle. In contrast, Sm expression was blocked in innervated muscle that overexpressed a dominant negative mutant of Ras (interferes with Ras signaling). This observation is relevant
Summary
Because NFAT and MEF2 have been implicated in the activation of slow muscle gene expression, a more com- plete knowledge of their downstream gene targets will be necessary for understanding their role in modulating fast and slow phenotype switches in response to various physiologic perturbations. Likewise, continued investiga- tions will also be required to fully elucidate additional downstream effectors (transcription factors) of CaN sig- naling and for a more complete picture of how CaN sig- naling integrates with other signaling pathways involved in both hypertrophic growth and phenotype transitions.
KEY POINT
All muscle skeletal muscle cells can mount a suitable physiologic response to chronic use. This action occurs via the integration of multiple intracellular signaling pathways and gene networks to modulate the activity of transcription factors and cellular proteins involved in protein synthesis and degradation.
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CHAPTER 5 The Muscular System: The Control of Muscle Mass 159 mouse skeletal muscle was also found to induce hypertro- phy and to prevent atrophy (29). In addition, constitutively active PI(3)K, the upstream activator of Akt, was also shown to prevent muscle atrophy (29). Further support for the involvement of the Akt pathway in muscle growth comes from transgenic gain of function experiments wherein skel- etal muscle restricted overexpression of constitutively active Akt1 resulted in enhanced growth of adult skeletal muscle (29). Conversely, in loss of function studies, mice carry- ing the targeted deletion of Akt1 (or Akt1 Akt2 ) showed growth retardation and a striking amount of muscle atrophy (30). In summary, activation of the Akt pathway led to activation of mTOR, which in turn activates its targets, p70 S6K , and inhibits PHAS-1/4E-BP1 (phosphorylated heat- and acid-stable protein-1; also known as 4E-BP1), which are necessary steps for increased protein synthesis and, thus, skeletal muscle hypertrophy.
to many exercise models and specifi cally to the overload model because MOV is associated with an increase in motor nerve activity (as measured by EMG signal) and increased Sm expression. It is interesting that several recent investigations have shown that NFAT transcription factors differentially translocate to the nucleus depending on the electrical stimu- lation patterns used, and that NFATc1, in particular, seems to act as a repressor of fast muscle gene expression in slow muscle (26,27). Additional research will be required to deter- mine if the increased motor nerve activity associated with MOV differentially modulates the NFAT transcription factors to facilitate a fast-to-slow fi ber type transition.
INSULIN-LIKEGROWTHFACTOR-1 (IGF-1)/PHOSPHATIDYLI-
NOSITOL-3-OH KINASE (PI(3)K)/AKT (PROTEINKINASE B)/
MTOR (MAMMALIAN TARGET OF RAPAMYCIN) PATHWAY
(IGF-1/PI(3)K/AKT/MTOR) IGF-1 has been shown to serve important functional roles during myogenesis and, in adult muscle, respond to muscle injury and growth-in- ducing stimuli such as MOV. In particular, one isoform of IGF-1 is upregulated in skeletal muscle only in response to mechanical stimuli and, thus, has been termed mechano growth factor (28). Studies on transgenic mice have shown that IGF-1 overexpression results in skeletal muscle hyper- trophy, and IGF-1 has also been shown to stimulate satellite cell proliferation which may contribute to its hypertrophic effect on muscle (Fig. 5.3).
Observations that IGF-1 has the potential to improve skeletal muscle regenerative capacity in injured, diseased ( mdx [muscular dystrophy]), and aged mice have prompted considerable interest in deciphering the downstream sig- naling pathway or pathways activated by IGF-1 leading to skeletal muscle hypertrophy. Briefl y, the signaling pathway downstream from IGF-1 binding to its membrane recep- tor (a receptor tyrosine kinase) involves the serial activa- tion of several downstream kinases (PI[3]K, Akt [cytosolic protein kinase recruited to the membrane when PI{3}K is activated; also known as PKB], mTOR, and p70 S6K ) leading to increased protein synthesis, a necessary step in the devel- opment of skeletal muscle hypertrophy (Fig. 5.3). Studies utilizing myogenic cells in culture have reported that IGF-1 signaling involves the calcium-activated CaN pathway (29);
however, the fi ndings of others dispute this observation by showing that treatment of cells with CsA did not block IGF-1-mediated hypertrophy of myotubes (29). Instead, the latter studies provided strong evidence for a PI(3)K-Akt- mTOR pathway by showing increased phosphorylation of these downstream IGF-1 effectors following treatment of cells with IGF-1 and by demonstrating that CsA treatment did not block this response nor could a calcium ionophore induce this response (29). The involvement of this pathway in inducing skeletal muscle hypertrophy was further sup- ported by animal studies wherein the levels of Akt phospho- rylation were found to be increased in response to MOV, and treatment with the mTOR inhibitor rapamycin nearly elimi- nated hypertrophy of the MOV-plantaris muscle (29). The overexpression of a constitutively active form of Akt in adult
KEY POINT
IGF-1 has been shown to serve important functional roles during myogenesis and, in adult muscle, respond to MOV.
MYOSTATIN Myostatin, or growth differentiation fac- tor-8 (GDF-8), is a member of the transforming growth factor- family, and like other members of this family, it is also secreted. The expression of myostatin is primarily restricted to muscle from early embryonic development to adult life. The targeted deletion of myostatin resulted in a highly muscled mouse phenotype that resembled a phenotype observed in two breeds of cattle (Belgian Blue and Piedmontese) as early as 1807 (31). This naturally occurring phenotype in cattle was described as an inher- itable condition of hyperplasia (increase in cell number) as opposed to hypertrophy (increase in cell size as seen with MOV) in 1982 and hence termed doubling muscling . A loss of function mutation in the human myostatin gene has also been shown to enhanced muscle growth. The fi nding that the absence of biologically active myostatin, whether due to a natural gene mutation (frameshift mutation due to 11-nucleotide deletion in cattle) or via gene targeting, led to increased muscle mass indicated that myostatin is a nega- tive regulator of muscle growth.
Because of myostatin’s obvious potential therapeutic value as a countermeasure of muscle-wasting diseases, studies have been undertaken to determine whether the absence of bio- logically active myostatin would be effective in maintaining muscle mass in several mouse models of muscular dystrophy, and whether the postnatal absence of biologically active myo- statin would induce skeletal muscle hypertrophy. Indeed, the inhibition of myostatin activity was found to have a favorable effect on maintaining muscle mass in dystrophic mice, and to induce a moderate level of skeletal muscle hypertrophic growth in postnatal skeletal muscle (32–36). As satellite cells
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160 SECTION 1 Exercise and Responses of Biologic Systems
Transcriptional control of skeletal muscle hypertrophy
An important aspect of understanding how skeletal muscle hypertrophy and phenotype are controlled undoubtedly involves the transcriptional regulation of muscle genes representing contractile proteins that constitute, in part, the protein accretion necessary for enlargement and for increased force development. Although the protein products of these muscle genes must be assembled in precise stoichio- metric amounts to produce sarcomeres, a common genetic regulatory program for controlling muscle gene transcrip- tion has not been identifi ed. An explanation may be derived from the following observations: (a) Most muscle genes are in different parts of the genome which may not be transcrip- tionally activated by the same signals; (b) although some promoter and enhancer regions of muscle genes share con- served DNA-regulatory elements (sites of protein binding, also called cis -acting element), not all muscle genes contain these regulatory elements within their control regions; (c) the existence of a conserved element within a gene’s control region does not necessarily imply a functional role for this element under all physiologic conditions (Fig. 5.4).
GENERALSKELETALMUSCLETRANSCRIPTIONALREGULATION
Although no genetic regulatory program comprised of a common set of cis-acting elements and trans-acting factors that regulates all muscle genes has been elucidated, recent are thought to be the primary cellular source of postnatal
muscle growth, the hypertrophic growth resulting from the postnatal absence of myostatin has been attributed to satellite cell proliferation and incorporation into existing fi bers. How- ever, new evidence suggests that postnatal skeletal muscle hypertrophy of wild type and dystrophic mouse muscle due to myostatin defi ciency can occur without the involvement of satellite cells (36). This fi nding is important because it indi- cates that the absence of postnatal myostatin may be an effec- tive countermeasure against disease-induced muscle wasting without depleting the satellite cell pool due to repetitive cycles of damage-induced degradation and regeneration. However, additional work will be required to confi rm this intriguing observation and to elucidate signaling components of the myostatin pathway to be used as targets to block myostatin’s negative infl uence on muscle growth.
TEST YOURSELF
Do the same signaling pathways regulate hypertrophic growth and phenotype shifts? Do you think there is cross talk between intracellular pathways so that hyper- trophic growth and phenotype shifts occur concur- rently? How does hypertrophic growth that is induced by MOV differ from that induced by a decrease in active myostatin in postnatal skeletal muscle?
−300 −170
Structural Gene Promoter
Regulatory elements Silencer Enhancer
5⬘
5⬘
βMyHC Control Region
3⬘ 3⬘ +1
+1 A
B
S E TATA 5⬘ UTR introns exons introns poly A E
E-box dMCAT EA/T-rich (−332/−312) (−290/−284) (−269/−258)
C-rich
(−240/−228) (−210/−203)
E-box/NFAT (−182/−171) NFAT TEF-1
SP1 TEF-1
TEF-1 dNRE-S
?
? ?
? Max
Parp
pMCAT
FIGURE 5.4 The modular nature of gene control regions is depicted. A. The general structure of a gene and the hypothetical location of regulatory regions that can infl uence its expression in a manner specifi c to tissue, developmen- tal stage, and perturbation. The modular arrangement of regulatory regions and elements allows for the integration of combinatorial signals in a gene-specifi c manner. B. A minimal human beta-myocin heavy chain (MyHC) promoter previously shown to direct the expression of a reporter gene in a pattern that mimics the expression pattern of the endogenous MyHC promoter throughout development and in response to mechanical overload. Numerous studies on the MyHC promoter have identifi ed the presence of a strong positive muscle-specifi c control region (300/170) that is highly conserved in sequence and location across species. The regulatory elements comprising this control region and their cognate binding factors are shown. This region contains highly conserved distal muscle-CAT (dMCAT;
290/284), A/T-rich (A/T-rich 269/258; also called GATA), C-rich (248/225, not shown: 160/140, 61/41), proximal MCAT (pMCAT; 210/203), and E-box/NFAT elements (182/171). In addition, a negative element resides immediately upstream from the dMCAT element and is termed negative regulatory element-sense strand (dNRE-S) (332/312). NFAT, nuclear factor of activated T-cell; SP1, specifi c protein1; TEF-1, transcriptional enhancer factor-1.
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