Lets Get Hypertrophic

Mechanisms of Exercise-Induced

Muscle Hypertrophy

Jason Cholewa

     Skeletal muscle is a postmitotic tissue and does not undergo significant cell replacement through the course of human life.  Cellular repair in skeletal muscle depends primarily on the dynamic balance between muscle protein synthesis and degradation (Toigo & Boutellier, 2006).  Increases in muscle CSA occur nearly exclusively through hypertrophy when protein synthesis exceeds that of protein breakdown (Brooks, Fahey, & Balwin, 2005c).  Skeletal muscle hypertrophy occurs via an increase in contractile proteins, increase in non-contractile intracellular volume, and an expansion of the extracellular matrix (Schoenfeld, 2010).  Myofibril hypertrophy, the addition of sarcomeres in parallel, accounts for the majority of skeletal muscle growth (Paul & Rosenthal, 2002).  Exposure of a myofiber to overload stimulates protein synthesis, leading to an increase in the size and volume of myosin and actin filaments, resulting in a greater number and volume of parallel sarcomeres, and thereby results in an increase muscle CSA (Toigo & Boutellier, 2006).

Sarcoplasmic hypertrophy occurs through increases in intracellular elements and fluid (Flück & Hoppeler, 2003), and has been suggested to lead to an increase in muscle CSA without significant increases in strength (Siff & Verkhoshansky, 1999).  Satellite cells (myogenic stem cells) located between the basal lamina and sarcolemma of the myofiber have been identified as responsible for mediating a significant portion of sarcoplasmic hypertrophy (Rosenblatt, Yong, & Parry, 1994).  When activated by mechanical or chemical stimuli, myogenic stem cells proliferate and fuse to existing skeletal muscle fibers, emptying their contents into the muscle fiber, and thus facilitating repair and growth (Toigo & Boutellier, 2006).   Myogenic stem cell fusion leads to skeletal muscle hypertrophy via two mechanisms.  First, myogenic stem cell fusion donates nuclei to the skeletal muscle fiber (Moss & Leblond, 1971), increasing the number of myonuclear domains, and enhancing mRNA expression throughout the muscle fiber (Toigo & Boutellier, 2006).  Second, myogenic stem cells express various myogenic factors that bind to the muscle gene promoter, thereby aiding in regeneration and growth (Sabourin & Rudnicki, 2000).

Cellular hyper-hydration (cellular swelling) is influenced by aniso-osmolarity, hormones, nutrients, and oxidative stress (Usher-Smith, Huang, & Fraser, 2009), and may also contribute to muscle hypertrophy beyond the acute increase in intracellular fluid by creating a more anabolic environment.  Cellular swelling has been shown to stimulate protein synthesis in mammary (Grant, Gow, Zammit, & Sheenan, 2000) and hepatic cells (Stoll, Gerok, Lang, & Häussinger 1992).  The increased pressure exerted against the sarcolemma with cellular swelling may pose a threat to cellular integrity, and thereby initiate a signaling response that results in the reinforcement of intracellular structures (Schoenfeld, 2011).  Häussinger (1996) hypothesized that cellular swelling is sensed by integrins and transduced down stream to signal protein synthesis, gene transcription, and inhibit proteolysis via mitogen activated protein kinase (MAPK).  Additionally, cellular hyperhydration may augment protein stimulus by enhancing amino acid uptake via phosphatidylinositol 3-kinase activation (Low, Rennie, & Taylor, 1997).

Performance of resistance exercise leads to an anabolic environment through mechanical and chemical signaling cascades.  An acute bout of resistance training results in the elevation of both anabolic and catabolic hormones and growth factors (Leite et al., 2011).  The metabolic stress and resultant accumulation of metabolites associated with resistance exercise has been suggested to promote anabolism by altering the hormonal milieu (Goto, Ishii, Kizuka, & Takamatsu, 2005), inducing cellular swelling, and promoting the expression of growth factors (Takarada et al., 2000).  Mechanical perturbations are sensed and molecularly transduced downstream to a number of pathways that couple myofiber contraction with chemical signals, shifting the intracellular protein balance to stimulate synthesis over degradation (Schoenfeld, 2010).  Additionally, sarcolemma deformation and myofibril damage during high degrees of muscle tension attracts macrophages and lymphocytes.  As macrophages phagocytize extracellular debris they produce cytokines which activate myoblasts and stimulate myogenic stem cell proliferation and differentiation, leading to a hypertrophic response (Vierck et al., 2000).

To better understand how betaine may improve body composition via muscle hypertrophy, downstream cellular signaling and the role of hormones in the hypertrophic response will be discussed.

Downstream Cellular Signaling

The cascade of downstream signals in skeletal muscle is specific to the modality of the exercise (Nader, 2006).  Resistance training activates a series of signaling cascades that promote gene expression and protein synthesis which over a series of consecutive bouts results in overall genotypic and phenotypic adaptations (Brooks et al., 2005b).  Initiation of messenger RNA (mRNA) translation has been identified as a regulating factor in protein synthesis (Bodine, 2006).  According to McCarthy and Esser (2010), the rate of protein synthesis is determined by both translational efficiency and translation capacity.  Translational capacity has been defined as the ribosomal content of a muscle fiber whereas the translation efficiency is determined by the amount of proteins synthesized per unit of RNA (Millward, Garlick, James, Nnanyelugo, & Ryatt, 1973).  According to Bassel-Duby, Rhonda, and Olson (2006) three primary anabolic signaling pathways have been identified: Mammalian target of rapamycin, mitogen-activated protein kinase, and calcium dependent pathways. The following is a brief review of each of the anabolic signaling pathways.

Mammalian target of rapamycin pathway.

The mammalian target of rapamycin (mTOR) is a member of the phsphatidylinositol kinase-related kinase family (Bodine, 2006) and integrates several upstream regulators of protein synthesis that are activated during resistance training (Miyazaki & Esser, 2009).  When phosphorylated, mTOR stimulates mRNA translation initiation by activating 70 kDa ribosomal protein S6 kinase (p70 S6K) via phosphorylation (Lawrence, 2001).  Concurrently, mTOR also hyperphosphorylates eukaryotic initiation factor 4E- binding protein 1 (4E-BP1), preventing the 4E-BP1 inhibition of eukaryotic translation initiation factor 4E (eIF4E) binding to the mRNA cap.  Collectively, mTOR regulates mRNA translation initiation via the activation of p70 S6K and inhibition of 4E-BP1 (Nader, 2006).

Specific to this review, growth factors such as insulin, growth hormone (GH), and insulin like growth factor-1 (IGF-1) stimulate protein synthesis via the mTOR pathway.  In brief, binding of insulin and IGF-1 to the respected receptor phosphorylates the insulin receptor substrate 1 (IRS-1), IRS-1 phophorylates phosphatidylinositol 3-kinase (PI-3K) which in turn recruits Akt from the sarcolemma (Bodine, 2006).  Similarly, the binding of GH to its membrane receptor activates janus kinase 2 (JAK2) which also activates PI-3K resulting in Akt recruitment (Spiering et al., 2008).  Activated Akt phosphorylates mTOR which continues the signal to initiate mRNA translation as described above (Miyazaki & Esser, 2009).  Additionally, Akt stimulates glycogen synthesis by phosphorylating and subsequently inactivating the glycogen synthetase inhibitor glycogen synthase kinase-3 (GSK-3; Bodine, 2006).

A growth factor-independent pathway has also been identified in the activation of mTOR (Goodman et al., 2010).  Contractile loading is transduced via mechanoreceptors to activate phospholipase D (PLD) which in turn interacts with Rheb, generating phosphatidic acid and activates mTOR (Hornberger et al., 2006).  Amino acid availability also effects mTOR activation.  Vacuolar protein sorting 34 (Vps34) is an amino acid sensitive member of the PI-3K family that activates mTOR independent of the Akt pathway (Miyazaki & Esser, 2009).  Vps34 activity has been shown to increase following resistance training in response to an influx of amino acids (MacKenzie, Hamilton, Murray, Taylor, & Baar, 2009).  McCarthey and Esser (2010) suggested that the PLD/mTOR pathway in conjunction with amino acid availability is responsible for the immediate initiation of mRNA translation following resistance exercise.

Mitogen activated protein kinase.

Resistance exercise has been shown to activate the mitogen activated protein kinases (MAPK) in a tension dependent manner (Sherwood et al., 1999).  Once activated, MAPK signals downstream to three distinct pathways: extracellular signal-regulated kinases (ERK 1/2), p38 MAPK, and c-Jun NH₂-terminal kinase (JNK).  MAPK activation increases skeletal muscle hypertrophy by activating an array of mRNA transcription factors that modulate cellular metabolism and growth (Kramer & Goodyear, 2007).

In particular, p38 MAPK activates myocyte enhancer factor-2 (MEF2) and peroxisome proliferator-activated receptor-γ coactivator-1α  (PGC-1 α) resulting in enhanced glucose uptake and fatty acid oxidation (Wagner & Nebrada, 2002).  JNK, on the other hand, acts on the nucleus to initiate the rapid increase in mRNA of the early response genes c-jun, MyoD, Myc, and c-fos, leading to cell proliferation, DNA repair, increased fast myosin heavy chain type II production, and cellular regeneration (Karin & Gallegher, 2005; Meissner et al., 2007).

Calcium dependent pathway.

Upon motor neuron stimulation the sarcolemma is depolarized, resulting in the release of Ca²⁺ from the sarcoplasmic reticulum, triggering cross-bridge cycling, and the subsequent contraction of the muscle fiber (MacIntosh, Gardiner, & McComas, 2005).  The Ca²⁺-dependent protein phosphatase calcineurin (CN) has been identified as a critical modulator of skeletal muscle hypertrophy and regulator of fiber-type specific genes (Chin et al., 1998; Dunn, Burns, & Michel, 1999).  Allen, Satrorius, Sycuro, and Leinwand (2001) demonstrated that CN targets MEF2 and nuclear factor of activated T cells (NFAT) leading to an increase in type II myosin heavy chain synthesis.  Additionally, CN has been shown to down regulate the myostatin gene (Michell, Dunn, & Chin, 2004), an inhibitory regulator of skeletal muscle hypertrophy (McPherron, Lawler, & Lee, 1997).

Hormones and the Hypertrophic Response

Resistance training results in both acute and chronic increases in several anabolic hormones, including testosterone, growth hormone, and IGF-1.  Additionally, the interaction of insulin, catecholamine and cortisol also influence the regulation of hypertrophy (Kraemer & Ratamess, 2005).  An elevated concentration of anabolic hormones will facilitate muscle growth by increasing the potential for receptor interactions, thereby stimulating protein synthesis, gene expression, and/or satellite cell proliferation and differentiation (West, Burd, Staples, & Phillips, 2010).

Specific to the scope of this review, the effects of insulin, insulin like growth factor-1, and growth hormone will be discussed.

Insulin.

Plasma insulin levels decrease during resistance exercise (Kraemer & Ratamess, 2005); however, the slight elevation in insulin associated with post exercise amino acid and carbohydrate ingestion significantly increases protein synthesis and reduces protein catabolism (Wolf, 2000).  Insulin increases skeletal muscle protein synthesis by affecting both translation efficiency and capacity.  Interaction of insulin with the receptor immediately increases mRNA translation efficiency via PI-3K/Akt/mTOR pathway (O’Brien, Streeper, Ayala, Stadelmaier, & Hornbuckle, 2001).  Occurring subsequently after mRNA translation initiation, insulin receptor activation increases the translation capacity of the muscle fiber by inhibiting ribosomal degradation and stimulating ribosomal DNA transcription (Ashford & Pain, 1986).  Additionally, insulin stimulates the uptake of selected amino acids such as leucine (Biolo, Delcan Flemming, & Wolf, 1995), which has been indicated as a rate limiting substrate in the initiation of mRNA translation (Norton & Layman, 2006).  Based on the anabolic effects of insulin in the presence of amino acids, the ingestion of carbohydrates and amino acids immediately following resistance training has been shown to increase muscle CSA over the course of several months (Gibala, 2000).

Although insulin significantly stimulates anabolism (Kraemer & Ratamess, 2005), insulin resistance may negatively affect adaptations in skeletal muscle induced by resistance training (Holten et al. 2004).  Paturi et al. (2010) demonstrated that insulin resistance resulted in a 25% lower soleus CSA hypertrophy in rats following 8 weeks of overload training.  The researchers reported that mTOR phosphorylation and upstream Akt phosphorylation were reduced by 30% in insulin resistant rats compared with controls.  Kata et al. (2010) also reported reduced hypertrophy and decreased mTOR phosphorylation following overload training in insulin resistant rats.  Because there were no changes in myogenic regulatory factors, Katta et al. concluded that insulin resistance may decrease the ability of skeletal muscle to activate mTOR and hypertrophy in response to increased loading.

Insulin like growth factor-1.

Insulin like growth factor-1 (IGF-1) is a peptide hormone of structural similarity to insulin, provides a primary anabolic response systemically (Schoenfeld, 2010), and displays enhanced effects in response to resistance training (Hameed et al., 2004).  Sarcolemma IGF-1 receptor binding stimulates myofiber protein synthesis via the PI-3K/Akt/mTOR pathway (Bodine, 2006).  Additionally, IGF-1 signals myogenic stem cell proliferation and differentiation by upregulating MyoD expression and fusion via M-cadherin adhesion molecules (Cornelison & Wold, 1997).

Plasma IGF-1 values rise approximately 6-9 hr following resistance training (Kraemer et al., 1993), reach peak values 16 hours post training (Chandler, Byrne, Patterson, & Ivy, 1994), and have been shown to be higher at rest in trained vs. untrained men (Rubin et al., 2005).  Systemically circulating IGF-1s are classified into two subtypes, IGF-1Ea and IGF-1Eb.  Circulating IGF-1s are synthesized in the liver and regulated by GH (Frystyk, 2010).  Generation of IGF-1 by GH is sensitive to changes in nutrition, such that GH receptor responsiveness is decreased in a fasted state, whereas increased delivery of insulin via the portal vein increases GH receptor availability on hepatocytes (Leung, Doyle, Ballesteros, Waters, & Ho, 2000).  Further, insulin increases IGF-1 bioavailability by suppressing hepatic IGF-1 binding protein (IGFBP-1) production (Brismar, Fernqvist, Wahren, & Hall, 1994).

Skeletal muscles express local IGF-1 genes coding for IGF-1Ea, IGF-1Eb, and also IGF-1Ec, also known as mechano growth factor (MGF) since it is activated by mechanical stimulation (Scheonfeld, 2010).  In response to mechanical loading, the IGF-1 gene is immediately spliced toward MGF expression and then transiently splices back toward IGF-1Ea and IGF-1Ba expression.  Local IGF-1 levels remain elevated significantly post training and have been shown to exert myogenic effects up to 72 hr following exercise (McKay, O’Reilly, Phillips, Tarnopolsky, & Parise, 2008).  According to Hill and Goldspink (2003), MGF secretion initiates the myogenic stem cell activation, whereas the transient increase in systemic IGF-1 up regulates myofiber protein synthesis required to complete cellular repair.

Growth hormone.

Growth hormone is secreted by the pituitary, and is regulated by the hypothalamus either positively or negatively via GH releasing factor (GHrF) and somatostatin, respectively (Giustina & Veldhuis, 1998).  GH is released during exercise as a result of elevated metabolite concentrations (lactate, CO₂, H⁺) and neural signals from higher brain centers, such as the motor cortex (Kraemer & Ratamess, 2005).  The magnitude of plasma GH elevation depends on the volume, intensity, and recovery periods during the exercise.  Specifically, resistance exercise employing higher volumes, moderate to high intensities, and shorter recovery periods between sets results in the greatest GH response (Frystyk, 2010).  GH impacts muscle hypertrophy in several ways.  First, GH binding to the receptor stimulates the translation initiation of protein synthesis via the JAK2/Akt/mTOR pathway (Spiering et al., 2008).  Second, GH positively affects body composition by inducing mobilization of triglyceride (TAG) for use in skeletal muscle growth (Vierck et al., 2000).  Third, GH stimulates skeletal muscle uptake and incorporation of amino acids in proteins (Hartman, Veldhuis, & Thorner, 1993), thus contributing to increases lean body mass.  Fourth, GH activates myogenic stem cells by inducing MGF mRNA expression in mechanically loaded skeletal muscles (Imanaka et al., 2008).  Finally, GH stimulates collagen protein synthesis leading to the hypertrophy and strengthening of muscle fascia and tendons (Doessing et al., 2010).