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Muscle fibers and properties
Skeletal muscle is composed of multinucleated, elongated contractile cells called myofibers or muscle fibers. Muscle fibers have a diameter between 10 and 100 μm, and a length, which can reach 30 cm (Wheater and Burkitt 1987). Each myofiber contains bundles of protein filaments called myofibrils that extend the entire length of the cell. A myofibril is composed of a chain of sarcomeres, which is the functional unit of contraction (Figure 2). A sarcomere contains the myosin, which forms the thick filament, and the actin, which forms the thin filament. Thin and thick filaments slide past each other during muscle contraction and each sarcomere short-ens to 70% of its uncontracted, resting length (Darnell, Lodish et al. 1990). Each muscle fiber is surrounded by a plasma membrane and an outer basement membrane (basal lamina). Dur-ing development, multinucleated muscle fibers are formed by the fusion and differentiation of mononucleated cells. Satellite cells are localized between the plasma membrane and the basal lamina of the muscle fiber and are believed to represent a muscle stem cell population capable of cell division for muscle growth and repair ((Campion 1984); (Blau, Dhawan et al. 1993); (Cornelison and Wold 1997)).
Figure 2: The skeletal muscle sarcomere. Schematic diagram showing the organization of the skeletal muscle sarcomere. Elastic titin filaments (gray) extend from Z-disc to M-band to form a stable, yet flexible myofibril scaffold holding myosin thick filaments (purple). Actin thin filaments (green) are capped at the Z-disc by Cap-Z (cyan) and cross-linked to other actin filaments in adjacent sarcomeres by the actinin (blue).
Contractile properties
The molecular basis of a muscle contraction can be described by the “sliding filament” theory. Two types of filaments are involved in this process, the thick filaments (myosin) and the thin filaments (actin). In the presence of ATP and calcium, actin filaments slide along myosin fil-aments thus allowing the shortening of the muscle necessary for a complete muscle contrac-tion. The detailed process is depicted in (Figure 3).
The speed at which a muscle fiber contracts depends on its metabolic and contractile proper-ties. There are two types of muscle fibers, the type I and type II fibers. Muscle fibers type I have a metabolism that is based primarily on the aerobic respiration. Their contractions are slow but fatigue resistant and therefore able to support long-term moderate activity. They are mostly found in the postural muscles. Muscle fibers types II are divided in mouse into IIA, IIB and IIX. Like the fiber type I, muscle fiber types IIA contain many mitochondria and my-oglobin. However, they are less resistant to long fatiguing activity as compared to type I fi-bers. Muscle fiber types IIB cause rapid, short and powerful contractions. They operate under anaerobic conditions and are needed during short intense exercise.
The contractile properties of muscle fibers and thus their classification are based on the meas-urement of the ATPase activity of myosin (Brooke and Kaiser 1970) or on the separation of myosin heavy chain isoforms by electrophoresis ((Schiaffino, Gorza et al. 1989); (Gorza 1990); (Duris, Renand et al. 1999)). The type I fibers (slow) and type IIA, IIX and IIB (fast) express MyHC isoforms I, IIa, IIx and IIb, respectively (Table 1). In addition to these adult myosin isoforms, other isoforms have been identified in the foetus: embryonic, fetal and al-pha-cardiac MyHC ((Weydert, Barton et al. 1987); (Picard, Gagniere et al. 1995); (Gagniere, Picard et al. 1999)).
Metabolic properties
The muscle fibers can also be distinguished according to their metabolic type (oxidative ver-sus glycolytic). There are two major types of fibers: oxidative fibers (type I fibers) and glyco-lytic fibers (type II fibers) (Table 1).
The measurement of enzyme activities is often used to determine the metabolic type of mus-cle fibers; for example, the activities of the isocitrate dehydrogenase (ICDH) or the succinate dehydrogenase (SDH), two mitochondrial enzymes of the Krebs cycle, are used to character-ize the aerobic oxidative metabolism of the muscle fiber. The measurement of the lactate de-hydrogenase (LDH), an enzyme that catalyzes the conversion of pyruvate to lactate during anaerobic glycolysis, is used to characterize the anaerobic glycolytic metabolism ((Robelin, Picard et al. 1993); (Gagniere, Picard et al. 1999)). The oxidative capacity of the muscle fibers has an inverse relationship with the diameter of the muscle fiber (Cassens and Cooper 1971).
Glucose and lipid metabolism of skeletal muscle
Mitochondria are specialized organelles and function as the energy center for the cell, which makes them essential for the survival of eukaryotic cells. They show significant differences between the heart muscle and skeletal muscle, both in their oxidative capacity ((Ogata and Yamasaki 1985); (Ventura-Clapier, Kuznetsov et al. 1998)) and the nature of their preferred substrates (Bahi, Koulmann et al. 2004).
Each mitochondrium consists of four major compartments that differ in composition, activity and function: [1] An outer membrane that is permeable to molecules with a molecular weight of less than 6 kDa, separates the mitochondria from the cytoplasm. [2] An inner membrane delimits the [3] intermembrane space from the [4] mitochondrial matrix, which contains various enzyme complexes of different metabolic pathways such as the Krebs cycle, the respiratory chain and the beta-oxidation of fatty acids.
The outer mitochondrial membrane
It contains multiple copies of a transport protein called porin, which forms together with the adenine nucleotide transporter (ANT) a large aqueous channel through the lipid bilayer. This membrane is permeable to molecules of up to 10,000 daltons, including small proteins and short chain fatty acids. Porin is also called the ‘Voltage-dependent anion channel’ (VDAC). It is the main channel for metabolites that regulate the mitochondrial respiration. In addition, several enzymes of various metabolic pathways may be associated with the outer membrane through interaction with porin. As examples, the hexokinase, the glycerolkinase, and the acyl-CoA synthetase (ACS) regulate mitochondrial function via interaction with the VDAC (Brdiczka, Kaldis et al. 1994).
The inner mitochondrial membrane
With reduced and selective permeability, the inner membrane constitutes a barrier for main-taining a concentration gradient of ions and metabolites between the intermembrane space and the mitochondrial matrix. Because of its low permeability, the inner membrane has many transport systems for ion exchange and metabolites. The transport of the substrates is carried out by more or less complex systems.
The anaerobic metabolic pathway in muscle
Under conditions of reduced oxidative capacity (insufficient amount of mitochondria or lack of oxygen), glycogen/glucose is converted into pyruvate by anaerobic glycolysis (Figure 4). The pyruvate is then converted into lactate in the cytosol in the presence of the enzyme lactate dehydrogenase (LDH). This process also leads to the reoxidation of the coenzyme NADH2 to 2H+ and NAD+. The energy balance of anaerobic glycolysis is relatively poor, because it leads to the net production of only two ATP molecules per molecule glucose.
Figure 5: The glycolytic pathway: In the case of insufficient oxygen, the cell produces energy by fermenta-tion (glycolysis) to operate the system. This is much less effective than the oxidative catabolism way, and the substrates are only partially degraded. In the presence of oxygen, mitochondria oxidize organic substrates producing energy directly used by the cells and releasing CO2.
The mitochondrial β-oxidation
Once the acyl-CoA from fatty acids (FA) are transported into the mitochondria through the carnitine shuttle, a series of transformations gradually and repeatedly removes two carbon atoms at each turn of the cycle. This repeated processing is called β-oxidation and produces one molecule of FADH2, of NADH2 and of acetyl-CoA at each cycle of acyl-CoA degradation (Figure 6).
The Krebs cycle consists of a series of biochemical reactions, whose purpose is to produce the reduced equivalents (NADH2 and FADH2) to be used in the production of ATP by the respira-tory chain. This reaction chain forms a cycle since the last metabolite, the oxaloacetate, is also involved in the first reaction. It is the final common pathway of all acetyl-CoA molecules, whether resulting from glycolysis or the -oxidation (Figure 7).
The term oxidative phosphorylation (OXPHOS) is employed to describe the coupling between the oxidation of the reduced equivalents and phosphorylation of ADP. This process involves five complexes with multiple subunits, which are located at the inner membrane and form the respiratory chain. Oxidation of NADH2 at Complex I (NADH:ubiquinone reductase) and of succinate/FADH2 at Complex II (succinate:ubiquinone reductase) releases the electrons, which are transported along the electron transport chain first onto ubiquinone (Q), then toComplex III and from there to cytochrome C (CYTC). Complex IV (Cytochrom C oxidase)then transfers the electrons onto molecular oxygen:
Complex I Q Complex III CYTC Complex IV O2 and Complex II Q III CYTC Complex IV O2
The electron transfer is achieved through a series of oxidation-reduction reactions with vari-ous constituents of the complex, which is coupled to the translocation of H + from the matrix into the innermembrane space (only through complexex I, III, and IV) The final electron ac-ceptor is oxygen, which is reduced to water by complex IV.
The electrochemical gradient of H + generates a potential difference on both sides of the inner membrane (ΔΨ =-220mV) and constitutes a source of electrochemical energy for the synthe-sis of ATP powered by ATP synthase or complex V (Figure 8).
The PPAR signaling and metabolism
PPARs (peroxisome proliferator activated receptors) are transcription factors belonging to the nuclear receptor family. PPARs form heterodimers with retinoid X receptors (RXR), and through the assistance of co-activators such as PGC1α (peroxisome proliferator-activated re-ceptor coactivator 1α), they regulate gene transcription (Figure 9).Three isoforms of PPAR exist in mammals, which differ in biological function and tissue distribution: PPARα, PPAR and PPARδ (also called PPAR ).
The PPARα isoform is expressed in tissues with high metabolic flux, such as the heart and liver. The isoform is expressed in various tissues, such as the adipose tissue. PPARδ, how-ever, is ubiquitously present in the body, with a significant level of expression in skeletalmuscle. PGC1α and PPARδ have a synergistic action and interact physically with each otheras shown by immuno-precipitation ((Wang, Lee et al. 2003); (Dressel, Allen et al. 2003)).
When PPARδ is overexpressed in muscle, muscle fibers transform into a more oxidative phe-notype ((Lunde, Ekmark et al. 2007); (Luquet, Lopez-Soriano et al. 2003)) with increased oxidative capacity of glycolytic muscle ((Luquet, Lopez-Soriano et al. 2003); (Wang, Zhang et al. 2004)).
Resistance to the obesity
Overexpression of a constitutive active form of PPARδ resulted in mice resistant to obesityduring a high fat diet program (Wang, Lee et al. 2003). In contrast, mice deficient in PPARδshowed a decreased basal metabolism, decreased heat production, and glucose intolerance(Lee, Olson et al. 2006).
Targets of the PPARs
The PDK4 gene has been identified as a direct and specific target of PPARδ (Degenhardt, Saramaki et al. 2007). In addition to this direct activation by PPARδ, there exists also an indi-rect activation. It has recently been shown that FoxO1 is a specific target of PPARδ (Nahle, Hsieh et al. 2008) and that FoxO1 in turn up-regulates the transcription of PDK4 (Furuyama, Kitayama et al. 2003). The signaling pathway PPARδ FoxO1 PDK4 may therefore cre-ate a positive feed-back loop and enhance the transcription of PDK4.
The kinase PDK4 phosphorylates the PDHc thereby decreasing its activity (Constantin-Teodosiu, Baker et al. 2009). Thus, activation of PPARδ is able to inhibit PDH by activating the transcription of PDK4. However, the metabolic impact on the oxidation of pyruvate has so far not been investigated.
Regulation of muscle mass
The maintenance of muscle mass is dependent on different signaling pathways that control the balance between the processes leading to atrophy and hypertrophy. Atrophy is characterized by a decrease in fiber diameter and increased protein degradation. In contrast, hypertrophy is characterized by an increase in size of muscle fibers and an increase of protein synthesis.
Muscle atrophy
Muscle atrophy or muscle loss can occur during pathophysiological conditions such as aging and cancer. It is characterized by a decrease in fiber diameter and number of cytoplasmic or-ganelles such as nuclei and mitochondria. Muscle atrophy is characterized by a decrease in the number of myonuclei by apoptosis ((Allen, Roy et al. 1999); (Ferreira, Neuparth et al. 2008)). Such unbalance of the nucleocytoplasmic ratio impairs the balance between protein synthesis and proteolysis in favor of a decrease in protein synthesis. During muscle atrophy, the signal-ing pathways are deregulated towards proteolysis (Figure 10) with subsequent activation of the different enzyme systems necessary for this function, which are: the proteasome ubiquitin pathway-dependent ubiquitin-ligases MuRF1 (Muscle Ring Finger protein 1) and atrogin-1/MAFbx (Muscle Atrophy Fbox) ((Bodine, Latres et al. 2001); (Gomes, Lecker et al. 2001)).
The FoxO pathway
In skeletal muscle tissue, there are three isoforms of FoxO transcription factors (Forkhead box O), FoxO1, FoxO3 and FoxO4. This family of transcription factors plays a crucial role in pro-tein degradation. Their function is inhibited by their phosphorylation via the Akt pathway.
Once phosphorylated, the FoxO proteins are excluded from the nucleus and cannot activate the expression of a number of target genes involved in muscle atrophy (also called atrogenes) such as atrophin-1/MAFbx and MuRF1.
Activation of FoxO proteins via their dephosphorylation occurs during muscle atrophy if the signaling pathways such as the PI3K/Akt (Phosphatidyl Inositol 3 Kinase) are deregulated (Stitt, Drujan et al. 2004). Overexpression of FoxO3 causes muscle atrophy (Sandri, Sandri et al. 2004). The role of FoxO3 is inhibited by the expression of PGC1α, a member of the super-family of nuclear receptors acting as transcription factor of target genes (Sandri, Lin et al. 2006).
FoxO1 is also involved in the loss of muscle mass (Kamei, Miura et al. 2004). Transgenic mice overexpressing this factor have a body weight lower than that of control mice. FoxO1 is a negative regulator of the expression of genes encoding the structural proteins of type I fi-bers, such as the slow myosin isoform, the slow troponin isoform and the α-tropomyosin, which leads to impaired skeletal muscle function (Kamei, Miura et al. 2004). In C2C12 my-oblasts, the activation of FoxO1 decreases the mTOR (mammalian Target Of Rapamycin) pathway, which is involved in the control of protein synthesis and increases the expression of 4EBP1 (eukaryotic initiation factor 4E binding protein 1). 4EBP1 is a negative regulator of initiation of translation and its upregulation thus leads to a decrease in protein synthesis (Southgate, Neill et al. 2007).
The NFκB pathway
Activation of NFκB (nuclear factor kappa B) pathway is implicated in several pathophysio-logical conditions characterized by the loss of muscle mass. Indeed, the activation of NFκB in mice induces ubiquitin-dependent proteolysis and significant overexpression of the ligase MuRF1 but not atrogin-1/MAFbx (Figure 10) (Cai, Frantz et al. 2004). Consistent with these data, it was reported that NFκB knockout-mice are resistant to atrophy secondary to the ex-pression inactivation of atrogenes like atrogin-1/MAFbx and MuRF1 (Hunter and Kandarian 2004).
The Myostatin/BMP pathway
The growth factor myostatin (which will be detailed in chapter III) also contributes to muscle atrophy. Durieux and collaborators induced ectopic expression of the myostatin gene after electrotransfer into the tibialis anterior muscle of adult rats (Durieux, Amirouche et al. 2007). They reported that overexpression of myostatin causes a significant decrease in muscle mass, 26fiber cross-sectional area and muscle protein content. No decrease in the number of fibers was found. The overexpression of myostatin has also caused a significant decrease in the expres-sion of genes encoding muscle structural proteins (e.g. MyHCIIb, troponin I and desmin), as well as a decrease in the expression of MyoD and myogenin. Myostatin inhibits Akt phos-phorylation and consequently induces an increase in active FoxO1. This activates the expres-sion of related atrophy genes (McFarlane, Plummer et al. 2006). A recent study identified a critical role for the BMP pathway in adult muscle maintenance, growth and atrophy. The au-thors showed that inhibition of BMP signaling causes muscle atrophy, abolishes the hyper-trophic phenotype of myostatin deficient mice and strongly exacerbates the effects of dener-vation (Sartori, Schirwis et al. 2013).
Table of contents :
1. CHAPTER: THE SKELETAL MUSCLE
1.1 General introduction
1.2 Structure of muscle tissue
1.3 Muscle fibers and properties
1.3.1 Contractile properties
1.3.2 Metabolic properties
1.4 Glucose and lipid metabolism of skeletal muscle
1.4.1 The anaerobic metabolic pathway in muscle
1.4.2 The aerobic metabolic pathway in muscle
1.4.3 The PPAR signaling and metabolism
1.4.4 Targets of the PPARs
1.5 Regulation of muscle mass
1.5.1 Muscle atrophy
1.5.2 Muscle hypertrophy
1.6 Muscle regeneration
1.7 Response of skeletal muscle to repetitive muscle damage
2. CHAPTER: DUCHENNE MUSCULAR DYSTROPHY
2.1 History and disease description
2.2 The DMD gene
2.3 Mutations of the DMD gene
2.4 Dystrophin and the Dystrophin Associated Protein Complex
2.5 Dystrophin isoforms
2.6 The Dystrophin Associated Protein Complex
2.7 Animal models for DMD
2.7.1 The mdx mouse
2.7.2 The GRMD dog
2.7.3 The HFMD cat
2.8 Revertant fibers
2.9 Treatment strategies for DMD
2.9.1 Gene therapy
2.9.2 The exon skipping
2.9.3 Cell therapies
2.9.4 The pharmacological approach
3. CHAPTER: MYOSTATIN
3.1 Myostatin gene and protein structure
3.2 The myostatin knockout mouse model
3.3 Myostatin expression
3.4 The myostatin signaling pathway
3.5 Regulation of myostatin activity
3.5.1 Molecules binding myostatin
3.6 The function of myostatin
3.6.1 Myoblast cell proliferation
3.6.2 Myoblast cell differentiation
3.6.3 Muscle cell regeneration
3.6.4 The role of myostatin in adipogenesis
3.6.5 Contractile phenotype
3.6.6 Muscle metabolism
3.7 Therapeutic strategies based on myostatin blockade
3.7.1 The different approaches to induce myostatin blockade
3.7.2 Myostatin blockade as a therapy against DMD
