Abstract 5. Chapter 1: Introduction Background Specific Aims Instrument Assisted Soft Tissue Manipulation 10

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3 Abstract 5 Chapter 1: Introduction Background Specific Aims Instrument Assisted Soft Tissue Manipulation Acetaminophen Supplementation and 8 Wk of Aerobic Exercise α7β1 Integrin Expression Following 56 D of Spinal Cord Injury 13 Chapter 2: Literature Review: The Role of the Focal Adhesion in Skeletal Muscle Introduction Integrins Structure and Activation Integrin Activation and the Role of Talin Talin and Muscular Load Kindlins: the Co-Activators Sarcomere Assembly Muscle Differentiation Myogenic Regulatory Factors and the α7 Integrin α7 Integrin Alternative Splicing and Localization β1 Integrin Alternative Splicing and Localization The α7β1 Integrin and the NMJ Genetic Disruption of the α7β1 Integrin Compensation of the α7β1 Integrin During Muscular Dystrophies The α7β1 Integrin Response to Muscle Loading 39 2

4 Muscle Precursor Cells FAK FAK Structure and Activation The Role of FAK in Muscle Development FAK and the Response to Loading Neurological Input and FAK in Skeletal Muscle FAK and the PI3K Pathway csrc Structure and Activation csrc in Striated Tissue Acetaminophen APAP and Skeletal Muscle APAP and Tendon Function Conclusion 60 Chapter 3: Methods IASTM and the α7β1 Integrin Pathway APAP Supplementation and 8 wk of Aerobic Exercise SCI and the Focal Adhesion 71 Chapter 4: Results IASTM and the α7β1 Integrin Pathway APAP Supplementation and 8 wk of Aerobic Exercise Soleus Gastrocnemius SCI and the Focal Adhesion 99 3

5 Chapter 5: Discussion 102 Bibliography 109 Appendix A: Raw Data 122 IASTM and the α7β1 Integin Pathway 123 APAP Supplementation and 8 wk of Aerobic Exercise 138 SCI and the Focal Adhesion 158 4

6 Abstract Introduction: Integrins are membrane-spanning heterodimers that connect the extracellular matrix (ECM) with the interior of the cell. They are capable of detecting signals from the ECM and initiating changes within the cytosol and vice versa. The most prominent integrin in skeletal muscle is the α7β1 integrin. It connects the muscle fiber to laminin and can direct signaling through focal adhesion kinase (FAK), a prominent tyrosine kinase that responds to sarcolemmal loading. In principle, FAK can initiate the activation of multiple muscle hypertrophy pathways. FAK expression and activation can be modulated by external stretch from massage and exercise and blunted with unloading. Methods: Three distinct experiments were conducted to understand the role of the α7β1 integrin pathway and its response to differing muscle loads. The first project investigated the role of instrument-assisted soft tissue manipulation (IASTM) and the α7β1 integrin response. Eleven healthy males participated in the study. Each received approximately 9 min of IASTM over their gastrocnemius muscle. Muscle biopsies were taken pre-treatment and 24, 48 and 72 h post-treatment. The second project studied the role of acetaminophen (APAP) supplementation and aerobic exercise on the integrin pathway in both the soleus and gastrocnemius. Twenty-four male Wistar rats were randomly assigned to four groups: sedentary +placebo (SED+PLAC), sedentary+apap (SED+APAP), exercise+placebo (EX+PLAC) or exercise+apap (EX+APAP). Rats in the APAP groups were given 200 mg/kg body weight with PLAC receiving equal volumes of saline solution. Rats in the exercise group exercised 5 d/wk for 8 wk at 20 m/min at an 8 grade. Exercise duration was gradually increased to 60 min/ session. The third project investigated the role of spinal cord injury (SCI) and its affect on the α7β1 integrin pathway. Twenty male Wistar rats were used in this study. They were randomly 5

7 placed into two groups, an SCI or sham injury (SHAM) group. SCI rats received complete transection of the spinal cord at T4 while SHAM had only the spinous process removed from the vertebrae. The gastrocnemius was removed from the rats 56 d post-surgery. Results: For the first project there were no significant interactions for time and IASTM or differences in individual time points. For the APAP project, there were no significant interaction effects for drugs and exercise. APAP supplementation increased expression of the 70 kda α7 integrin, total and phosphorylated FAK and csrc, and total p70s6k in the soleus independent of exercise. There were no exercise effects in the soleus. In the gastrocnemius there were no interaction effects for any of the proteins investigated. APAP supplementation decreased phosphorylated p70s6k. There were no significant exercise effects, however there were large mean increases in FAK phosphorylation and ERK1/2 levels. In rats with SCI, a large but not significant increase in the expression of the α7 integrin was seen. There was a significant decrease in total and phosphorylated FAK and phosphorylated p70s6k. Conclusion: External manipulation of skeletal muscle via IASTM has no affect on the α7β1 integrin pathway up to 72 h post-treatment. APAP supplementation can induce hypertrophic signaling in a fiber-type dependent manner, possibly through the focal adhesion and independent of exercise. SCI induces decreases in mechanotransduction signaling in the cell through FAK although there is an strong but not significant indication for an increase in α7 integrin expression. 6

8 Chapter 1: Introduction 7

9 1.1 Background Skeletal muscle is a plastic, terminally differentiated tissue that is responsible for carrying out motor movements. It s the major metabolic reserve of amino acids and is responsible for the majority of whole body metabolism. Muscle responds to its environment in a stimulus-dictated manner, meaning that chronic loading through exercise such as resistance training will increase muscle size, strength and muscular endurance and chronic aerobic exercise that focuses on a continuous, rhythmic dynamic movement, such as cycling or jogging, will produce increases in metabolic and mitochondrial-related proteins. These changes, regardless of the type, are almost universally considered to be beneficial for muscle tissue and organismal health. The absence of exercise or complete unloading from injury or disease will decrease muscle health. A prominent point of convergence for detecting these stimuli are integrins. Integrins are heterodimeric proteins that span the cell membrane, connecting the extracellular matrix and cytoplasm (86). In skeletal muscle the prominent integrin is the α7β1 integrin. It binds laminin in the extracellular matrix and connects it to a conglomeration of proteins called the focal adhesion (25). The absence of the α7 integrin promotes progressive myopathies (79) with changes mainly affecting the myotendinous junction (114). The α7β1 integrin can protect cells from muscle damage (19), rescue mice from muscular dystrophies (18; 27) and can respond to eccentric exercise (105; 181). Integrins themselves have no enzymatic or kinase capabilities. Following integrin ligand binding and activation the signal is transferred to focal adhesion kinase (FAK), which becomes activated 8

10 by phosphorylation of Y397 (134; 135). FAK is mainly a sarcolemmal and costameric-based protein that recruits proteins to the focal adhesion, acts as a scaffolding protein and acts as the major kinase of the adhesion (75). FAK responds to overload (56; 70), unloading (50; 70; 101), massage (48) and exercise (164). FAK relays its signal through two potential hypertrophic pathways: csrc (150) and phosphatidylinosital-3 kinase (PI3K) (40). Acetaminophen (APAP) is a commonly used over-the-counter analgesic used to treat general pain and muscle soreness. Acute supplementation of APAP blunts the protein synthesis response following acute resistance training (156) but increases muscle size and strength following chronic resistance training (152). APAP has also been shown to decrease tendon function in humans (35) and to decrease tendon collagen crosslinking and stiffness in rats (37). The role of tendon health and force translation of the tendon makes the α7β1 integrin and its downstream signal relayers an interesting target. Spinal cord injuries create debilitating conditions that can cause muscle atrophy. No study has investigated the role of spinal injuries and integrins in skeletal muscle but unloading caused by whole body polyneuropathy alters costameric proteins, including the α7β1 integrin (3). The response of these pathways to exercise in striated tissue has been well documented. However, the role of FAK-mediated changes is less documented. The following projects are designed to expand the literature concerning the α7β1 integrin and its downstream effectors to various muscular stimuli. 9

11 1.2 Specific Aims Loading of skeletal muscle has profound effects on muscle performance, cell health and overall organismal well being. Investigating the role of passive massage through instrument-assisted soft tissue manipulation (IASTM), aerobic exercise and acetaminophen supplementation, and spinal cord injury should give a wide view into the effects of differential muscle stress. The forces that get transmitted through the mechanosensory proteins of the muscle should signal a protective and adaptive response. No study has investigated the role of the α7β1 integrin following IASTM, acetaminophen supplementation, or following spinal cord injury. The purpose of these investigations is to understand the molecular changes seen in skeletal muscle following these various interventions and provide possible explanations has to why these interventions are causing molecular changes. There are three projects that will be investigated. The first study will look at the role of IASTM and the response of the α7β1 signaling pathway over the course of 72 h following treatment. The second project will investigate the role of acetaminophen supplementation and aerobic exercise and possible changes in the α7β1 integrin pathway and markers of muscle hypertrophy. The final study will seek to determine the role of 56 d of muscular unloading caused by induced spinal cord injury and potential changes in the mechanosensory proteins of skeletal muscle Instrument Assisted Soft Tissue Manipulation 10

12 Instrument-assisted soft tissue manipulation (IASTM) is an often used preventative and rehabilitative technique. The technique consists of a trained practitioner using specialized stainless steel tools that are stroked over the target area with applied pressure. Manual massage of the vastus lateralis has been shown to increase activation of FAK immediately after massage (48). Specific Aim #1: To determine if a routine IASTM technique, Graston Technique, can cause changes in the α7β1 integrin pathway activation following treatment. We hypothesize that IASTM will not change the activation of the α7β1 integrin pathway. We will test this hypothesis using SDS-PAGE and Western immunoblotting. Specific Aim #2: To elucidate whether there are increases in the expression of proteins of the α7β1 pathway over 72 h We hypothesize that there will be no changes in the total expression of any of the proteins of the α7β1 integrin pathway following IASTM at 24, 48 or 72 h post-treatment. We will measure this by SDS-PAGE and Western immunoblotting Acetaminophen Supplementation and 8 Wk of Aerobic Exercise Acetaminophen (APAP) is a commonly used over-the-counter whole body pain reliever. Constant supplementation has been shown to be beneficial for skeletal muscle health (152) but 11

13 deleterious for the tendon (35; 37). No evidence exists in following administration of APAP and the α7β1 integrin or the focal adhesion. Changes in the extracellular matrix components following APAP supplementation may be promising for inducing changes in the mechanosignaling properties of skeletal muscle. Aerobic exercise and the role of chronic aerobic exercise has been shown to increase FAK expression and activation (164) but the authors did not investigate potential changes either upstream or downstream of FAK. This study should clarify the role of chronic aerobic exercise and the α7β1 integrin signaling pathway. Fiber type differences of the components of the focal adhesion have been well characterized (54; 57; 70). There is potential for fiber-type dependent changes based upon APAP supplementation and aerobic exercise. Specific Aim #1: To determine if there is an acetaminophen (APAP) and aerobic exercise interaction on components of the α7β1 integrin pathway following 8 weeks of APAP administration and exercise training There is no evidence in the literature that has provided sufficient evidence that APAP can interact with the integrin complex in skeletal muscle. We hypothesize that there will be no APAP and exercise effect on the α7β1 integrin. We will measure this by SDS-PAGE and Western immunoblotting. 12

14 Specific Aim #2: To compare main effect differences between the APAP and exercise groups. We have pilot data that shows 5 wk of aerobic exercise training with or without APAP administration does not cause changes in expression of the α7β1 integrin. We hypothesize that there will be no change between the APAP group and their activity level control. Furthermore, we expect that expression of FAK and ERK1/2 will increase following 8 wk of exercise training independent of APAP administration. We will measure this by SDS-PAGE and Western immunoblotting. Specific Aim #3: To determine differences in the α7β1 integrin and focal adhesion response in varying fiber types Type 1 muscle fibers have more quantity of integrin and focal adhesion proteins compared to type 2 muscle fibers. We hypothesize that any changes seen in either intervention will occur in the type 1 soleus compared to the type 2 gastrocnemius. This is based upon the motor recruitment of the exercise protocol and the aforementioned fiber type-dependent quantity of these costameric proteins. We will measure this by SDS-PAGE and Western immunoblotting α7β1 Integrin Expression Following 56 D of Spinal Cord Injury The role of the integrin response following myopathies has been well documented (3; 27; 73; 77; 79). However, there has been no investigation looking at the response of the focal adhesion 13

15 following long-term unloading caused by spinal cord injury. FAK is very susceptible to decreases in load (50; 56; 70; 101) and these decreases happen relatively quickly, as soon as 1 d in some cases. Specific Aim #1: To investigate changes in the focal adhesion, α7β1 integrin and hypertrophic proteins 56 d following spinal cord injury. We hypothesize that there will be a decrease in FAK expression and activation and downstream hypertrophic proteins. We further hypothesize that there will be no decrease in the α7β1 integrin. We will measure this by SDS-PAGE and Western immunoblotting. 14

16 Chapter 2: Literature Review: The Role of the Focal Adhesion in Skeletal Muscle 15

17 2.1 Introduction Skeletal muscle is a major component of the mammalian body. It is responsible for gross motor movements such as walking and running and is a very potent metabolic resource. Because of its major roles in the human body, skeletal muscle has been shown to be incredibly plastic to external stresses such as exercise, both aerobic and anaerobic, and partial or complete unloading. One often used manipulator of the skeletal muscle stress responses is resistance training. The principal skeletal muscle response to resistance training, provided adequate nutrition is available, is increased protein synthesis. This increase in protein synthesis increases total muscle crosssectional area by driving the expression of the major contractile components of the sarcomere, mainly myosin and actin. In a protective mechanism, trained skeletal muscle responds to loading by being more resistant to cellular damage compared to untrained muscle. One line of thinking is the proteins of the α7β1 integrin pathway respond to this overload to protect the muscle fiber. This review will cover the overall biochemical profiles of the focal adhesion. Brief structural summaries of relevant proteins will be followed by physiological data related to their function in muscle and other relevant tissue will be discussed. 2.2 Integrins Structure and Activation Integrins are non-covalently bound type 1 heterodimers that exist in all cell types and are responsible for coordinated signals from the extracellular matrix (ECM) to the cytosol and vice versa. Generally, the signal for integrin activation occurs from the cytosol in an inside-out manner that protects the cell from constitutively activated integrins. There are 18 known α 16

18 subunits and 8 β subunits that combine to form 24 distinct integrins (86). While it had been presumed that there were large (140 kda) glycoproteins responsible for binding fibronectin or laminin in the ECM to the cytoplasm, their proposed structure and uniform name remained unproposed until 1986 (146). Tamkun and colleagues were able to identify the primary sequence and suggest a name to unify the literature. They chose integrin, as these large glycoproteins were integral membrane proteins and necessary for ECM and cytoplasmic integrity (146). This seminal study further suggested that the structure of the integrin has the N-terminal portion of the protein largely in the ECM (90%) with a relatively tiny C-terminal domain through the membrane and in the cytosol (10%). To date, there is relatively sparse data regarding the structural biochemistry of integrins but the data published have given substantial information regarding their function. After the Hynes laboratory uniformly proposed the primary sequence, researchers began to investigate the tertiary and quaternary interactions between the integrin subunits. Many biochemical assays had proposed that integrins had a low affinity and high affinity ligand binding conformation. The nature of integrins suggested that they are generally inactive in resting cells and respond quickly to a stimulus. One study aimed to use Forster resonance energy transfer to determine if the change in conformation could be seen by changes in fluorescence as measured by flow cytometry. By placing immunospecific fluorotags that bound only to the platelet specific αiibβ3 integrin, it was seen that large extracellular changes occurred in the integrin conformation with and without the presence of fibrinogen, its preferred ligand (141). This large spatial change needed to be clarified as the directionality of this switch from low 17

19 affinity to high affinity remained unclear. More insight was gained following a study using degradation and cleavage patterns of the αiibβ3 integrin. Using purified αiibβ3 integrin in a solution that contained an arginine endoproteinase in a serum with no Arginine-Glycine- Aspartate (RGD) sequences (a prominent integrin binding sequence) yielded two products of 80 kda and 55 kda. By mimicking the previous experiment but adding an RGD sequence, they obtained three cleaved products: an 185 kda, 85 kda and 30 kda. These alternative cleavage sites suggested that an arginine is protected in the RGD-null serum of the β3 integrin subunit. By mapping where these arginines were, the researchers concluded that this cleavage site was in a suspected loop region that interacts with the α subunit. Thus, a major shift was occurring from the interface, indicating that the ligand interactions may be between the heads of the integrins (33). The mechanisms behind the transition between the inactive and active positions remained vague. Beginning in the mid-1990 s, atomic-level crystal structures were beginning to propose solutions to how integrins interacted with their ligands. Lee and colleagues (98) were the first to provide strong atomic-level evidence that the high and low affinity states were driven by large extracellular conformational shifts (approximately 10 Å). In the I-domain of the α integrin, a major predicted ligand binding domain, this is driven by differences in metal ions in the metalion-dependent adhesion site (MIDAS). In α subunits without the I-domain, the β integrin has a similar fold that can dictate ligand binding. The authors conclude that this preference towards metal binding is a major component as to whether the integrin is active (bound with Mg 2+ ) or inactive (bound with Mn 2+ ). Continued structural data of various parts of the integrin extracellular domains were published but it wasn t until 2001 that a crystal structure of a near complete integrin was completed. In the structure for the αvβ3 integrin that contained everything 18

20 but the transmembrane domain (TMD) and cytosolic tails, the domain structure of both subunits was refined. In the α subunit, the N-terminal side has a β-propeller domain that makes up a large portion of the globular head. This is followed by a thigh domain which is linked to the calf-1 domain by a small linker domain. Attached to the calf-1 domain is the calf-2 domain which would connect to the transmembrane portion of the integrin. The β subunit is a bit more complex. At its N-terminus, it has a βa domain that interacts with the propeller domain of the α integrin. Attached to the βa domain is a hybrid domain, which is attached to 4 EFG-like repeats before it binds to the tail domain (170). Using their active structure, they were able to predict that the β subunit goes through a massive shift between the EFG domains from its inactive state to active (Fig. 1). Xiong and colleagues furthered their research by growing a crystal of the αvβ3 integrin while attached to an RGD ligand. In their structure they noticed that the ligand was bound between both subunits and made heavy contact between each interface. Their data also reinforced their previous data that suggested that the α subunit changes its orientation far less in the presence of ligand compared to the β subunit (171). The most complete data available for the integrin structure was completed by Zhu et al. They were the first to confirm the bent state in crystal form and provided negatively-stained electron microscopy images of the transformation of an inactive to active integrin (176). Further, they added their crystal positions to a molecular dynamics program and simulated cytosolic force production and integrin reaction. They saw that intracellular filamental actin dynamics were largely responsible for the structural shift in the integrin (176). 19

21 Figure 1. Transformation of the integrin from its non-ligand bound inactive form to its active state. Adapted from (170). The switch from an inactive to active state is mainly driven from an inside-out signal. This signal propagation, whether it comes from antigen binding, ligand binding, etc., depends on a complex signaling cascade that causes the initial step of integrin activation: the separation of the tails in the cytoplasmic domain. The first evidence that the binding between the tails conferred the activation state of integrins was demonstrated by truncation of the cytoplasmic domains, creating a constitutively active integrin (120). Hughes et al. expanded upon this by creating point mutations throughout the cytoplasmic domains of the αiibβ3 integrin. They demonstrated that disruption between a well conserved 6-8 amino acid sequence on each integrin hindered a salt bridge between the two tails (84). These mutations increased not only ligand affinity but also the activation of the downstream signal relaying protein focal adhesion kinase (FAK). The TMD also has a substantial effect during inactive stabilization. Insertion of a cysteine residue into adjacent TMD positions in both the α and β subunits created a disulfide bond that restricted the αiibβ3 integrin into the inactive state (107). The use of leucine substitution for each glycine in each 20

22 subunit TMD increased integrin affinity and ligand binding by decreasing the transmembrane helix interaction (106) Integrin Activation and the Role of Talin Talin is a 270 kda protein that binds to the β integrin and links it to the actin cytoskeleton and vinculin, a scaffolding protein of the focal adhesion (5). Discovered in 1983 in chicken gizzard smooth muscle, it was shown to be localized to the membrane by fluorescence microscopy (29). There are two main isoforms of talin, talin 1 and talin 2. Talin 1 is widely expressed throughout the body and talin 2 is mainly restricted to striated tissue. In the mammalian genome, both talins are very similar, but distinct, and may be redundant (5). For the context of this section, talin will be discussed in general unless specified. Talin is composed of an N-terminal globular head with a large, C-terminal rod that is attached by a thin linker domain that is easily accessible to calpain-ii cleavage (31). The important binding characteristics of talin are mainly relegated to the 4.1 band, ezrin, radixin, moesin (FERM) domain, a cloverleaf-shaped domain that often interacts with the plasma membrane (31). While no single structure of talin has been completed, all domains have been determined by X-ray crystallography and have given a suitable working model. Talin works to activate integrins by using the head domain to directly bind to the β subunit of the integrin complex. By using selective mutations in both the talin head and the β1d integrin, it was determined that the cytoplasmic portion the β integrin has a specific talin head binding site (32). Furthermore, isolation of the talin head from the rod domains was enough to activate integrins 21

23 (32). Talin was also found to be essential for inside-out integrin activation. Small interfering RNA (sirna)-induced talin knockdown decreased integrin activation (144). More experiments from this study also showed that mutation of certain residues of the β tail overcame the sirna knockdown, furthering the evidence that it is the tail of the integrin that is responsible for insideout activation Talin and Muscular Load Muscular load has a large role in altering the costamere in skeletal muscle. The integrin and talin roles have been implicated as quite important in cell protection (the integrin changes are discussed in detail below). Talin s role in skeletal muscle has been investigated, although not heavily. The earliest evidence for talin in skeletal muscle was the discovery that it was localized to the neuromuscular junction (NMJ) (137), heavily located in the myotendinous junction and, to a lesser extent, regular intervals in extrajunctional regions of muscle (148). Talin is responsive to load as 10 d of unloading, followed by 2 d of resuspension, increases talin protein expression and talin mrna expression increased in the MTJ as measured by in situ RT-qPCR (61). Cyclic strain placed upon C2C12 myocyte cells increases talin protein and mrna expression as well (61). Eccentric exercise increased talin expression in the plantaris (type II dominant fiber) by approximately 2-fold while only having a slight impact on the soleus. This upregulation was generally found in the MTJ (60). Talin 1 and talin 2 have different roles in skeletal muscle. Myoblasts contain talin 1 while talin 2 doesn t appear until the cells become differentiated. Talin 2, when placed into undifferentiated 22

24 myoblasts, cause instability within the actin cytoskeleton. Furthermore, talin 2 is preferentially located at the costamere in developed muscle suggesting that talin 1 connects integrins to actin in motile, undifferentiated muscle and talin 2 develops a more stable connection in mature fibers (138). Mouse skeletal muscle that had talin 1 knocked-out still developed detectible focal adhesions and high integrity sarcolemmas. However, talin 1 was necessary in maintaining those focal adhesions at the MTJ. Talin 1 knockouts develop progressive myopathies and severe decreases in isometric and eccentric force production (45). The same lab produced an elaborate model comparing the effects of deletion of talin 2 or both talins in mice muscle. Talin 2 knockouts developed myopathies similar to those seen in talin 1-deficient mice. Talin 2 knockouts had a higher percentage of centrally located nuclei at seven months in both the soleus and gastrocnemius but no sign of membrane damage. Talin 2 was not necessary for sarcomere formation but there was severe disorder and an increase in talin 1 at the MTJ, suggesting that talin 1 was upregulated and localized to the MTJ but was unable to stabilize it. Mice with both talins knocked-out had severe disruption in myoblast fusion and development, sarcomere formation and focal adhesion assembly (46) Kindlins: the Co-Activators Talin has been shown to work with a co-activating family of proteins called the kindlins. Kindlins are a family of three membrane-oriented proteins that interact with the focal adhesion. Its name is derived from Kindler syndrome, an autosomal recessive disorder that causes severe skin blisters and is the result of the missing kindlin-1 gene. Each member of this family contains a FERM domain and can bind to the β integrin cytoplasmic tail (90). Kindlin-2, the most widely 23

25 expressed of the kindlins, is necessary for integrin activation. Montanez et al. (118) used multiple models to investigate the role of kindlin-2 in the focal adhesion. In a kindlin-2 knockout model using mice, embryos disappeared following 7 d, suggesting that complete knockout of kindlin-2 is lethal, while 50% knockdown led to normal mice. In another model, a sustainable kindlin-2- null embryonic stem cell line was established. These knockout cells had severely reduced cellular adhesion to various laminins compared to wild-type. Furthermore, talin levels were unchanged between the two cell lines, suggesting that kindlin-2 may be an interacting protein between the integrin and talin. Point mutations in the integrin showed that the FERM domains of talin and kindlin-2 bind to different locations on the cytoplasmic β tail. This was an indication that kindlin may be able to activate integrins. However, over-expression of kindlin-2 did not increase integrin activation but co-expressing talin s FERM domain did, providing evidence that the talin FERM domain works along with kindlin-2 to activate integrins. How this interaction occurs is still uncertain but a model has been suggested. Kindlin-2 enhances integrin activation by binding a loop within its FERM domain to a cytoplasmic sequence on the β integrin tail distinct from talin s binding site. It is possible that kindlin-2 anchors itself to the membrane, reducing the flexibility of the β tail and allowing a more favorable binding position for the talin head (108) (Fig. 2). 24

26 Figure 2. Kindlin and talin function together to activate integrins. 1) Integrins are inactive, with their head domains facing the membrane. 2) Kindlin binds to its distinctive binding site on the β integrin cytoplasmic tail. This is not enough to confer integrin activation. 3) Talin binds to its specific binding site on the β1 tail. Alone, talin cannot promote efficient integrin activation. 4) Kindlin and talin binding initiates cytoplasmic tail domain separation and integrin activation. Model from (108) Sarcomere Assembly Integrins have been implicated in multiple roles in muscle development. Muscle develops when precursor muscle cells, myoblasts, fuse together creating multi-nucleated myotubes. This is followed by cellular adhesion to the ECM and other myotubes, which join together to form myofibrils. The myofibrils contain the basic functional unit of skeletal muscle, the sarcomere. The sarcomere is composed of α-actinin, which makes up the z-disk, and ultimately binds to the focal adhesion connected to the integrin. Myosin and the sarcomeric ruler, titin, connect to the z- 25

27 disk and are responsible for creating force during a muscle contraction. Cross-bridge cycling between filamentous actin (f-actin) and myosin causes sarcomere contraction and transduction of force through the z-line and into the costamere. This process is critical during muscle formation. Inhibition of calcium signaling and lack of mechanical stretch, either passive or electrically induced, in cultured muscle cells severely blunts sarcomere organization (51). Reassembly of these sarcomeres began following the application of electrical current and washing the cells of the calcium inhibitors (51). The role of the integrin during sarcomere assembly and initial myofibril stability is not well understood but it is known that the integrin has an important function and it most likely has to do with recruitment of proteins to the focal adhesion following z-disk formation. Measurements of single focal adhesions from isolated cardiac myocytes has shown that the cell promotes cellular adhesion and focal adhesion formation in accordance to the force placed upon the focal adhesion by actomyosin force production (10). This promotion is most likely based upon the ability of the integrin to bind to its ECM ligand as inhibition of integrin adhesion in electrically-stimulated C2C12 cells disrupts sarcomeric assembly (62). There is evidence that β1 integrin-null myoblasts can form competent muscle. Cultures of these β1-null myoblasts were able to display sarcomeres that were indistinguishable from wild-type cultures, although fusion was delayed approximately 10 d following that of the wild-type (78) Muscle Differentiation Integrins play a significant role during myofibrillogenesis outside of sarcomeric viability. During myofibrillar assembly, immunofluorescent staining has shown that proteins of the focal adhesion are the first to be organized into observable and repeated patterns (149). There is substantial 26

28 evidence that specific integrins and their alternative isoforms interact during the developing process. The α7β integrin is the major laminin-binding α integrin in developed muscle (25; 161) but is lacking during the initial moments of primary muscle development. Developing myoblasts contain multiple α subunits, principally αv, α1, α3, α4, α5, α6, α7, and α9 which all interact with the β1 subunit (113). Electrical activity of the muscle cell has been linked to not only sarcomeric assembly as mentioned previously, but muscle differentiation as well. Inhibition of electrical activity decreases costamere assembly and β1 subunit protein expression during multiple points of differentiation in muscle cell cultures (157). The α4 integrin has been implicated as being a major component during early muscle development by altering expression and location throughout the development cycle. In cultured mouse myoblasts, the presence of the α4 subunit was visible by immunostaining in 11 day-old embryos. By day 13, myotubes that had begun developing a basal lamina were heavily stained with the α4 integrin intramuscularly. By day 15, there was a continuous lamina and the α4 integrin began to appear in the cell surface. Secondary myotubes began to form at day 16 which resulted in a decrease in α4 integrin. This suggests that the α4 integrin is responsible for fusion of myoblasts into myotubes. This was authenticated by the inability of myotube formation following the addition of inhibitory antibodies against the α4 integrin (129). This is contrasted by evidence that chimeric α4-null embryonic stem cells form and develop into muscle and are readily identifiable in mature tissue, implying that the α4 isn t necessary during muscle development (172). Muscle staining using mouse muscle cultures shows the presence of the α5 integrin on myoblasts and newly formed myotubes and the stains tended to be along the 27

29 membrane (85). Interestingly, the α7 subunit is only seen on the sarcolemma of primary myoblasts following terminal differentiation and myotube formation but can be seen during all stages of secondary muscle fiber formation. This is most likely because of the lack of laminin during the initial steps of primary myotube formation, allowing for motility and fusion. The presence of laminin surrounding the primary muscle cell likely creates high affinity sites during secondary fiber formation (64). During muscle formation, the α7β1 integrin becomes heavily present at the myotendinous junction (MTJ). The α7 subunit is present at day 14 of chicken embryos in the MTJ with slight α7 staining along the sarcolemma only after day 20. Before day 14, the α3 integrin is the most highly present and is enriched at the MTJ. Furthermore, the alpha7 integrin is not present at the end of 21 d cultures, but the α3 integrin is (11). Injury of rat soleus by crushing was monitored over the course of 14 d. mrna analysis showed that two isoforms of the α3 integrin were present, α3a and α3b at 5 and 7 d post-injury, and mrna for β1 gradually increased expression from day 3 to day 14 (125). Genetically upregulating the α7 integrin in developing myoblasts and in developed muscle fibers improves overall cellular adhesion to laminin and cell proliferation as well as being resistant to apoptosis. All of these changes do not change differentiation and doesn t drastically change overall cell gene expression (103). The β1 subunit is necessary not only for proper muscle development, it is necessary for embryonic development. Genetic knockout of the β1 subunit is lethal almost immediately following implantation of chimeric embryos. Heterozygous β1 chimeric embryos develop normally and were similar to wild-type mice (55). Mutations to the cytoplasmic domain of the β1 integrin result is decreased recruitment and assembly of proteins of the focal adhesion (1). 28

30 Schwander et al. (136) have demonstrated that the muscle specific β1 subunit regulates not only sarcomere assembly but myoblast fusion and muscle differentiation. They reported that mice that lacked the β1 integrin died at birth. Analysis of embryos showed evidence that there were postural deformities caused by muscle defects. They further noted that the lack of β1 integrins resulted in short muscle fibers and the accumulation of unfused myoblasts in vivo caused dysregulation in the formation of the muscle cytoskeleton. Finally, they determined that integrins are necessary for cellular adhesion after the fusion of multiple myoblasts results in a shared sarcolemma. This evidence is contrary to previous literature that showed that the β1 integrin is not needed for myoblast and myotube formation but decreases the rate of fusion (78). A potential regulator of this differentiation switch is muscle integrin binding protein (MIBP) a cytoplasmic protein that can bind with the β1 subunit. As muscle develops into a laminin-rich environment, the integrin subunits change, as mentioned above. MIBP binds to the laminin binding α7bβ1 integrin, not the fibronectin binding α5β1 integrin, and decreases following terminal differentiation. Furthermore, overexpression of MIBP decreases myogenic differentiation and cell adhesion. This is related towards MIBP levels decreasing laminin deposition into the ECM (100) Myogenic Regulatory Factors and the α7 Integrin The expression of the α7 integrin has been linked to the presence of the myogenic regulatory factors (MRF), proteins which determine and differentiate developing muscle cells. The principle MRFs are MyoD, myf5, myogenin and MRF4. MyoD has been the most implicated in α7 29

31 integrin expression. MyoD, and to a smaller extent myogenin, can increase the expression of the α7 integrin by binding to its promoter site and MRF4 may increase α7 expression in a promoter independent manner (178). Support of this data is seen in a study measuring promoter activity. Using a promoter activity assay, Xiao et al. (169) reported that transfection of MyoD increased α7 promotor activity and this was blunted by the addition of c-myc, a strong muscle differentiation inhibitor. δef1, a potent inhibitor of muscle differentiation, shares a promoter binding site with MyoD. Overexpression of δef1 decreases MyoD binding and results in decreases in α7 integrin expression. Mutating this shared site rescues integrin expression (87). The previous studies are somewhat contrasted in mice with genetic knockouts of MyoD. While these mice had altered fusion rates and smaller and less nucleated myotubes, they had equivalent staining of the α7 integrin compared to wild-type cell (85) α7 Integrin Alternative Splicing and Localization The most prevalent and relevant α subunit in skeletal muscle is the α7 subunit. The α7 subunit is alternatively spliced and consists of multiple isoforms: 3 different cytoplasmic domain variants (α7a, α7b and α7c) (44; 142; 179) and 2 extracellular variants (α7x1 and α7x2) (179). These isoforms are developmentally regulated. The α7a isoform is more prevalent during differentiation, the α7b isoform during replication of myoblasts and the α7c is seen only following terminal differentiation (142). The α7b is the only form that is found in myoblasts and outside of muscle cells while the α7a and α7c are found only in differentiated muscle (80). In mature skeletal muscle, α7a and α7b are heavily localized at the MTJ and α7b and C are found in the belly of the muscle (Fig. 3). All three can be located in post-synaptic regions of the skeletal 30

32 muscle neuromuscular junction (NMJ) (111). Recent evidence has shown that the α7b is the most prevalent isoform and is heavily located in the costamere (3). Figure 3. Localization of the different α7β1 integrin isoforms in mature skeletal muscle. In mature mouse skeletal muscle, αx2 is the only isoform detected while L8 and C2C12 cells have equal amounts of αx1 and αx2 as measured by RT-PCR (179). It appears these isoforms have differing affinities towards extracellular ligands. α7x1 and α7x2 both bind collagen but only α7x2 efficiently bound laminin. The authors suggested that α7x1 is natively inactive as it was activated by addition of β1-activated antibodies and α7x2 is constitutively active (177). 31

33 These splice variants can be regulated during muscle recovery from injury. Following unilateral transection of the soleus in Wistar rats, the α7a isoform had a higher expression from 3 d postinjury until 28 d post-injury. At the next sampling period, 56 d post-injury, the α7b isoform had approximately 90% of the expression. The control rats expressed no α7a isoform (88). The extracellular isoforms changed as well. The control rats had approximately 60% α7x2 and 40% α7x1 expression. Each subunit had approximately 50% expression 2 d post-injury. Expression of the α7x1 isoform increased to 70% at 5 days post-injury, decreased to 40% at day 10 post-injury, and then maintained that expression level for the remainder of the sampling days (88). Evidence during transgenic over-expression of the α7bx2 integrin showed large increases in integrin concentrations at the sarcolemma but also isolated concentrations in the cytoplasm (103). During this over-expression, excess α7 integrin is located in endoplasmic reticulum (ER) (103) but can be rescued with an increase in β1d expression (104) β1 Integrin Alternative Splicing and Localization The β1 integrin is the most abundant β subunit, capable of binding up to 12 different α subunits and multiple extracellular ligands (12). There are four predominant β1 isoforms, β1a-d. In skeletal muscle, the β1a and β1d are the prevalent isoforms (159) with the β1d being restricted only to striated tissue (14). Diversification of the β1 subunit occurs by alternatively splicing the exon that encodes the cytoplasmic domain portion. The difference in the two is minimal but profound: the β1d has a unique 24 amino acid residue terminal compared to the 21 residue terminus of the β1a (174). Belkin et al. (14) did a comprehensive study of the β1a and β1d 32

34 subunits. They found that the β1d isoform is the most prevalent in striated tissue but is not found in replicating myoblasts. The predominant isoform during muscle differentiation is the β1a isoform. The expression of β1a begins to decrease upon myotube formation while β1d expression drastically increases. Furthermore, while the β1a appeared to be readily present across the majority of the sarcolemma, the β1d subunit was localized to focal adhesions and was responsible for activation of signaling cascades (14). The displacement of the β1a subunit by β1d is largely based on its association with cytoskeletal proteins. In measurements between cytoskeletal interactions using digitonin, the β1d was less soluble than β1a, suggesting β1d has a stronger interaction. Additionally, β1a tended to bind to α-actinin while β1d had a higher affinity for talin and the extracellular matrix. This higher affinity binding of the β1d subunit is thought to be necessary to maintain a stable structure that can transmit the force of a muscle contraction (13). Increased expression of the β1d subunit in C2C12 cells increased transcription of the α7 integrin gene, ITGA7, and the laminin α2 gene, LAMA2 (104) The α7β1 Integrin and the NMJ The clustering of acetylcholine receptors (AChR) on the post-synaptic area of the skeletal muscle NMJ has been linked to involvement of the basal lamina. A main inducer of AChR clustering is agrin, a protein of the basal lamina. Since laminin-1 is a major component of the basal lamina, it was hypothesized that the α7β1 integrin may also have a role in the proper orientation of the receptors. Indeed, both integrin subunits localize to AChR through the induction of laminin. Furthermore, distinct isoforms were located at these sites. The α7ax2 and α7bx2 were both found to co-localize at the AChR and immunoprecipitation provided evidence that the α7β1 33

35 integrin forms a complex with AChR in a laminin-dependent manner. Addition of anti-α7 antibodies can also induce AChR clustering in small concentrations and dissociate the complex in high concentrations without the presence of laminin or agrin (24). The exact manner of how the α7β1 integrin facilitated this interaction was later investigated. It was determined that laminin, in the presence of agrin, induced AChR clustering and α7β1 integrin NMJ localization more readily than either protein alone. Pre-incubation of laminin also lowers the amount of agrin needed to form AChR clusters while accelerating the formation. Interestingly, mutations in a cytoplasmic tyrosine of the α7ax2 and α7bx2 increased agrin-induced AChR clustering, providing evidence that phosphorylation of the this tyrosine may be a negative regulator of agrininduced AChR clustering (26) Genetic Disruption of the α7β1 Integrin Defects in the integrin gene have been shown to induce myopathies. Mayer, et al., (114) deleted the α7 gene (ITGA7) in mice to study its potential role during development. Surprisingly, they found that homologous deletion of ITGA7 led to viable mice that remained fertile, providing evidence that the α7 gene is not a necessary component of muscle development or there is a related compensation. However, the mice had muscle characteristics similar to progressive muscular dystrophy such as fiber necrosis, variable fiber size, fiber turnover, evidence of phagocytosis and centralized nuclei. The main defects found were mainly located in the MTJ. This was clarified in a study investigating the role of MTJ development in α7-null mice. Another α7-null mice model found that β1 expression doesn t change but more of the non-muscle specific β1a isoform is found (131). Miosge et al. (117) found that α7-null mice had less and smaller 34

36 interdigitations at the MTJ and a disorganized basement membrane. In a follow-up study using the same α7 knockout model, Nawrotzki et al. (119) found that in the MTJ, the β1d subunit colocalizes with the α5 integrin and its preferred ligand, fibronectin, was detected in high levels of the basement membrane. The authors conclude that a prevalent role in the α7β1 integrin is to gradually displace the α5β1 in the MTJ as the α7β1-laminin binding relationship is more robust. Out of 117 patients with unclassified congenital myopathies, 3 non-related patients had genetic changes to the α7 gene (77). Each individual had at least one distinct mutation. One individual had defects on both alleles: a splice mutation that caused the addition of 21-bp on one allele and a mutation causing a 98-bp deletion in the other. One individual had the same 98-bp deletion on one allele and a 1-bp frame-shift mutation on the other. The other patient had no discernible mutations but had markedly low levels of α7 mrna. In a cohort of 210 patients with myopathies of unknown etiologies, 35 had lower levels of the α7a and α7b integrin subunits. Further analysis showed that 6 of the 35 had no α7b levels and had progressive muscular dystrophies. Thirty biopsies of the 35 had enough sample leftover for genetic analysis and 7 polymorphisms were detected, all in the extracellular portion. Six of the polymorphisms were silent base changes and the other one was a histidine to arginine amino acid change, which was present in almost 50% of the chromosomes (123) Compensation of the α7β1 Integrin During Muscular Dystrophies The α7β1 integrin and focal adhesion complex form, along with the dystroglycan complex, part of the skeletal muscle costamere (Fig. 4). Individuals with Duchenne s muscular dystrophy (DMD) and Becker s muscular dystrophy (BMD) have increased expression of the α7 integrin as 35

37 measured by immunofluorescence and RNA analysis (79). The same study used a laminindeficient dystrophy (dy/dy) mouse model, a DMD analog model (mdx) and control mice and found that α7β1 expression was higher and that the ratio of developmentally regulated alternative splicing favored the undifferentiated state. The dy/dy mice had different levels of the intracellular spice ratios of the α7 and β1 subunits and mdx mice had differing levels of the extracellular splice ratios (79). The compensation of the α7β1 integrin produced a feasible hypothesis that increased expression may blunt the effects of DMD. In a dystrophin-utrophin knockout mouse model (mdx/utr), a model that mimics human DMD more accurately, the α7bx2 gene was genetically upregulated with the help of a muscle creatine kinase-driven promoter, a constituently active muscle gene, and expression levels were confirmed with immunofluorescence. The α7bx2 transgene resulted in increased expression of the β1d subunit, providing evidence that the α7 chain potentially regulates the β1 chain. The mdx/utr and mdx models had higher levels of the β1a subunit, suggesting that these models are in a more undifferentiated state, and β1a levels mirrored the wild-type mice in the α7bx2 mice. Perhaps more importantly, transgenic mice lived longer and were able to maintain muscle mass (28). Burkin and colleagues (27) further investigated this model with a focus on the mechanisms behind these integrin-related changes. The major implications of transgenic integrin expression in mdx/utr-deficient mice was a more stable MTJ, increased regeneration capacity through the recruitment of satellite cells and increased muscle fiber hypertrophy. There was also a decrease in cardiomyopathy, a symptom seen in mdx/utr-deficient mice (27). It appears that upregulation of the α7 integrin does not prevent dystrophy in mdx-deficient mice. Transgenic expression could only quadruple the concentration of α7 integrin and this was not enough to differ from the mdx- 36

38 deficient model. However, increased expression of the β1d chain prevented muscle damage in the mdx-deficient mice (104). Figure 4. Graphical representation of the muscle costamere. The costamere directs longitudinal forces and anchors the muscle fiber. Adapted from (16). Alterations in the Akt/mechanistic target of rapamycin (mtor)/p70s6k hypertrophic protein signaling pathway have also been implicated in regards to the α7β1 integrin. Integrin-linked kinase (ILK) binds to the β1 cytoplasmic domain and is enriched at costameres and the MTJ. Inhibition of ILK caused decreases in Akt activation (124) and deletion of the β1 subunit increases insulin resistance and caused an approximate 50% change in Akt phosphorylation in muscle tissue (180). Boppart et al. (18) demonstrated that increasing the α7bx2 integrin in 37

39 C2C12 cells initiates ILK binding to the α7 subunit and an increase in activation of the Akt pathway. Furthermore, the transgenic cells had decreases in apoptotic protein signaling and a trend for less apoptotic markers compared to the mdx/utr-knockout cells. The dystroglycan complex, the major laminin-binding component of the costamere, and the α7β1 integrin provide independent, although not preventatively redundant, roles. When both dystrophin and the α7 integrin subunit were knocked out, there was substantial muscle wasting and reduction in overall muscle health. The most apparent defects were in sarcolemma viability, the MTJ and muscle turnover (72; 128). Utrophin is another major laminin binding protein. It is similar in structure and function to dystrophin except it is located mainly at the NMJ and MTJ. To determine the importance and redundancy of utrophin, Welser et al. (163) knocked it out along with the α7 subunit in mice. They found that that the mice had mild and progressive dystrophies, an increase in dystrophin at the NMJ, decreased force production and muscular endurance, decreased MTJ viability, but no change in sarcolemmal integrity. Another type of muscular dystrophy, merosin-deficient congenital muscular dystrophy 1A (MDC1A), is caused by mutation in the laminin-α2 gene, disrupting binding of the major structural proteins of the muscle. The most severe end of the spectrum is a complete loss of the laminin-α2 protein and, thus, a severely weakened basal lamina. Unlike muscle dystrophies affecting specifically the muscle, MDC1A-stricken individuals have severe neuropathies. The lack of laminin-α2, either in total or in function, disrupts the formation of laminin-211 and -221, the most prevalent isoforms in the skeletal muscle basal lamina. Patients with MDC1A have lower levels of the α7β1 integrin (79). Over-expressing the α7bx2 gene increased the lifespan of 38

40 laminin-α2 knockout mice (53). Interestingly, the transgenic mice did not retain muscle mass but were better able to stop the loss in muscular force at 8 weeks of age. The mechanism behind this suggests that the α7β1 integrin regulates the composition of the ECM as transgenic mice had higher transcription levels of collagen and different α integrin subunits and lower transcription levels of disintegrin and MMP2, proteins that disrupt integrin binding (53). Human limb girdle muscular dystrophies (LGMD) are caused by mutations in the sarcoglycans, components of the DGC. There are four different isoforms of sarcoglycans and disruptions occurring in each respective isoform can form a different muscular dystrophy. In LGMD 2F, the δ sarcoglycan is mutated. Transgenic mice over-expressing the α7bx2 integrin did not have any notable effect on LGMD 2F mouse model. These mice had similar exercise capacities and MTJ stability. The α7bx2 upregulation didn t change protein localization and other components of the DGC appeared to be adequately bound to the basal lamina (116) The α7β1 Integrin Response to Muscle Loading Each integrin subunit has been shown to change expression during various types of muscle loading. Three days of disuse by hindlimb suspension in rats did not change protein or mrna expression of the β1 integrin. However, 12 h of reloading following disuse increased both protein and mrna expression (115). Expression of the β1 integrin subunit does not significantly change after 34 d of bed rest in humans but does increase after 3 wk of 3 sessions of 4x10 maximal coupled contractions on a flywheel ergometer. However, these levels returned to normal after 9 wk of training (101). Muscular inactivity caused by sensitive-motor polyneuropathy, a systemic 39

41 disease that ultimately creates nerve damage and muscle weakness, created higher levels of α7a integrin and decreased levels of β1d integrin compared to normal individuals (3). Exercise may be a means to activate integrin signaling or expression. Concentric contractions, caused by the shortening of the sarcomere by crossbridge cycling, have the potential to alter the integrin signal by displacing the force of the contraction laterally through the costamere (16). The role of ions, second messengers, protein signaling or other mechanisms that respond to skeletal muscle action potentials is another potential area of integrin regulation. It is possible that rhythmic changes in calcium concentration, sarcolemmal stretch, etc. can manipulate integrin and focal adhesion activation. The role of aerobic exercise that does not cause muscle damage, i.e. the predominant mode of exercise for most individuals, and the integrin response has not been investigated in skeletal muscle. The most often used research method dealing with integrins in skeletal muscle is through eccentric exercise. Eccentric exercise generates muscular force without the use of sarcomeric shortening. The force capabilities are a result of sarcolemmal stretch, which create passive interactions between actin and myosin, the rigidity of titin, and, where integrins may have a role, the binding capabilities between membrane proteins and the ECM. Thus increasing integrin expression may prevent muscle damage and sarcolemmal integrity by increasing the cumulative strength of the ECM/sarolemmal binding strength. Eccentric exercise through downhill running is the most prevalent research model. Transgenic mice expressing 8x the α7bx2 subunit as wildtype mice have decreased extracellular signalrelated kinase (ERK) 1/2 and Akt pathway activation compared to wildtype mice immediately and 3 h after 30 min of downhill running in mice. Transgenic mice had lower levels of muscle 40

42 damage. Wildtype mice had increased transcription of the α7b subunit 24 h post-exercise and elevated, although not significant, 1 wk post-exercise (19). The transcriptional regulation following 30 min of downhill running in mice showed that transcription of the α7 integrin subunit is approximately 5.5 higher 3 h post-exercise compared to other α subunits and transcription of the alternative isoforms are all higher 3 h post-exercise. These levels return to pre-exercise levels 24 h and 1 wk post-exercise (21). Another component of the study looked at the response of the MTJ and the integrin isoforms. The α7a integrin localized to the MTJ immediately after exercise and α7b co-localized with tenascin-c, a marker of muscle development or damage. α integrin-null mice have severe muscle damage after one bout of exercise, similar to wildtype. However, after a repeated bout one week later, α integrin-null mice had even more muscle damage while the wildtype had none, suggesting that the α7β1 integrin may have a role in preventing muscle damage in as little as one bout of exercise (21). We have recently reported (manuscript in review) that 90 min of downhill running causes the loss of the full length α7 integrin and that heat treatment 48 h before exercise may alleviate the loss of the 70 kda α7 subunit. Sixty min of downhill running in transgenic mice overexpressing the alpha7bx2 integrin prevents the loss in force production and macrophage appearance in skeletal muscle (105). Perhaps more interesting, transgenic mice had large fiber cross-sectional area 7 d post-exercise bout, increased mtor activation, higher levels of myogenesis as measured by embryonic myosin heavy chain and centrally localized nuclei in small muscle fibers. The authors also note that this is most likely satellite cell independent even though satellite cells were increased in the transgenic mice 1 d post-exercise (105). A follow-up study by the same lab used the same model but instead trained the mice 3x/wk for 4 wk. They found the exercised transgenic 41

43 mice had elevated levels of phospho-akt, mtor and p70s6k compared to wildtype sedentary and wildtype exercised as well as transgenic sedentary mice. There were no changes in α7b levels following training in the wildtype mice. They further reported that the exercised transgenic mice had larger muscle fibers and larger whole muscle cross-sectional area (181). Passive loading has been suggested as a means to induce hypertrophic signaling (20; 82). Passive loading via an external source, such as manual or tool-assisted massage, could increase the sarcolemmal stretch response, activating integrins and initializing downstream kinase signaling. Indeed, passive stretching of the sarcolemma via massage has shown to increase activation of the main protein downstream of integrins, FAK, in humans (48). This is most likely induced by the external stretch response. The internal loading of the costamere, caused by contraction of the sarcomere z-discs pulling on the focal adhesion complex, could possible initiate inside-out integrin activation. This is not likely as the stress of the massage treatment is unlikely to directly activate crossbridge cycling or alter ion flux across the sarcolemma enough to generate action potentials Muscle Precursor Cells Recent investigations have demonstrated the role of integrins in muscle differentiation from muscle precursor cells (MPC), such as satellite cells and mesenchymal-like stem cells (MSC), following injury. 42

44 Satellite cells are muscle specific stem cells that contain the paired-box transcription factor Pax7, and are generally referred to as Pax7 + cells. Pax7 is necessary to survive and for normal neural development. Mice that were heterozygous for Pax7 appeared normal and were fertile but Pax7 (-/-) developed to term, appeared normal, but 97% died within the first 3 weeks and those that did live were considerably unhealthy (109). The authors suggest that altered formation of the cephalic neural crest was the major cause in the physical abnormalities of these mice. Pax7 (-/-) mice that do survive to adulthood are far smaller than wild-type mice (although muscle fiber size did not appear to be affected) and have a severe loss in satellite cell number during postnatal development (121). Further, Pax7 seems to be necessary not for initial satellite cell proliferation, as there was no difference in markers of proliferation after 4 days between Pax7-null and wildtype mice, but absolutely necessary for long term proliferation and satellite and mature muscle cell maintenance (121). However, Lepper and colleagues (99) have shown that Pax7 is not necessary for adult satellite cells and there is age-dependent change in Pax7 from embryonic to fetal to postnatal to adult muscle. During satellite cell fusion to the myotube, the α4β1 integrin has been demonstrated to be upregulated and mrna of the α7 integrin subunit is increased in wild-type cells during satellite cell emergence (22). The α7β1 integrin is highly present in muscle stem cells (15; 122). The increase in α7 has been proposed as a mechanism to bind and organize the basal lamina surrounding the fiber (22). The process by which satellite cells bind and integrate into existing muscle fibers supports this. The α7β1 integrin is heavily located on the basal, or non-muscle fiber, side of the satellite cell. This creates a strong tether to laminin that allows the apical side, the side in contact with the muscle fiber, to allow for proper cellular communication (25). This asymmetric binding allows for the proper regulation of the satellite 43

45 cell population by changing myf5 expression in neighboring satellite cells, creating committed and uncommitted progenator cells (95). Muscle MSC are multipotent cells that reside in skeletal muscle but do not contain Pax7. There is evidence that the appearance of MSC as a result of eccentric exercise may be α7 integrin dependent. Sedentary transgenic mice overexpressing the α7bx2 integrin had equal amounts of MSC as eccentrically exercised wildtype mice, approximately 9%, as measured by fluorescenceactivated cell sorting. This number corresponds to double the sedentary wildtype. Exercised transgenic mice had 18% of cells detected as MSC. α7-null muscle only contained approximately 2% both in sedentary and exercised conditions. Injection of extracted MSC from the alpha7bx2 transgenic mice into wildtype mice was associated with increases in embryonic myosin heavy chain and stimulation of Pax7+ satellite cells post-eccentric exercise (158). 2.3 FAK The roles of talin and kindlin indicate they are the major proteins responsible for integrin activation but neither of these proteins is capable of conducting this signal to the cytosol. How integrin activation gets translated into intracellular changes mainly falls under the responsibility of FAK. FAK is a 125 kda non-receptor tyrosine kinase that triggers signaling cascades and acts as a scaffolding protein in the focal adhesion (74). The main line of research that has looked into FAK is within cancer cells, where FAK controls cell life, motility and proliferation. Arguably the most important function of FAK in these cells is preventing anoikis, a programmed cell death that prevents cancer metastasis. In cardiac and skeletal muscle, FAK presents itself as a major 44

46 mechanosensor, responsible for relaying the signal from the α7β1 integrin and activating prosurvival/hypertrophy signaling pathways. The major investigations into FAK began with the discovery that induction of cancer with a retrovirus containing the protein csrc increased the phosphorylation of a 120 kda protein (89) and this phosphorylation increased in multiple cells types. This 120 kda protein was shown to co-localize to the focal adhesion via immunofluorescence microscopy and was thus termed focal adhesion kinase (134). Further elucidation of FAK s role came when it was determined that integrins relay their activation signal through FAK. Using monoclonal antibodies that can induce integrin activation and clustering, it was noted that gradual increases in phosphorylation of a 130 kda protein matched that with the known time course of integrin clustering (94). Additionally, the phosphorylation signal increased with increasing levels of antibody. There remained a conflict in the literature as to whether the 120 kda and 130 kda proteins were actually FAK. Guan and Shalloway provided concrete evidence that these unidentified proteins were indeed FAK (71). They purified and isolated the unidentified proteins and matched them to csrc and β1 integrin-activated cell lines. They then put them through immunoprecipitation meant to detect FAK and their results clearly showed that this phosphoprotein that had been identified as either a 120 or 130 kda protein was indeed FAK FAK Structure and Activation The structure of FAK shares the characteristics of other focal adhesion and membrane-bound proteins, such as talin and kindlin, as it has an N-terminal FERM domain. This is linked to the 45

47 regulatory kinase domain, which is followed by the C-terminal focal adhesion targeting (FAT) domain (Fig. 5). Fig. 5. Regulatory domains of focal adhesion kinase (FAK). The main activation site is located in a linker domain at the tyrosine 397 residue. Phosphorylation of Y397 recruits phosphatidylinosital-3 kinase (PI3K) and csrc. Binding of csrc activates the kinase domain of FAK at Y576/577, conferring full activation of FAK. Figure based on (59) The FERM domain is largely responsible for the autoinhibitory state of inactive FAK. This is because the FERM domain can interact with the FAT domain to prevent access to the main FAK phosphorylation site, Y397. The first study to identify this used truncated FAK in insect cells. They found that deletion of the certain residues in the FERM domain increased FAK activation. Using glutathione-s transferase (GST) bound peptides that mimicked sequences from the FAT domain, they found the complex bound full length FAK as well as multiple truncated versions that didn t contain an FAT domain. They were also able to replicate their results in vivo by demonstrating that expressing a truncated FERM domain increased cell cycling (47). The FERM domain is also capable of binding to the tail of β integrins (134). 46

48 Between the FERM and kinase domains is a linker domain that contain the main activation site for FAK. Activation occurs following phosphorylation and it was discovered by Schaller and colleagues (135) that phosphorylation of Y397 occurs via autophosphorylation. This was achieved by using trypsin cleavage on recombinant FAK in wild-type cell types and in e. coli that were devoid of protein tyrosine kinases. The peptide cleavage for phosphorylated FAK match between the cell types, showing that FAK is autophosphorylated (135). Importantly, mutation of Y397 to F397 abrogated csrc binding affinity and lost a majority of FAK s signaling capabilities (135). The importance of csrc will be discussed in a later section. Lietha et al. (102) carried out a seminal study detailing how FAK is stabilized during its autoinhibitory state. By crystallizing a near complete FAK protein in the inhibited state, they discovered that the FERM domain makes contact with the kinase domain, burying the Y397 within a groove in the FERM domain. The other major phosphorylation sites of FAK, Y576 and Y577, reside in the kinase domain and are protected by the FERM domain and a cleft formed by surrounding helices of the kinase domain. In this study they also tested potential activating mechanisms. They found phosphorylation of Y576 and Y577 prevented the FERM domain from contacting the kinase domain, thus preventing inactivation, and that initial displacement of the FERM domain is the important step in activating FAK (102). Once the FERM domain moves, Y397 is quickly phosphorylated and this causes a conformational change in the kinase domain allowing Y576 and Y577 open to activation by csrc (102) (Fig. 6). 47

49 Figure 6. Activation of FAK. Inactive FAK is caused by autoinhibition from the FERM domain binding the Y397 residue and preventing it from moving from inside the protein complex. Disruption of this binding, from either small molecules or integrin activation, opens Y397 to the cytosol where it is quickly autophosphorylated and partially active. This prevents autoinhibition and opens up an activation loop containing Y576/577 on the kinase domain, allowing for phosphorylation by csrc and complete kinase activation. The FAT s main role is locating FAK to focal adhesions. This domain can bind to synthetic peptides mimicking β1 integrin tails (133). More likely, however, is the FAT domain binds to talin, positioning itself near the cytoplasmic tails of the integrin complex and in a position to relay the activation signal while also anchoring the adhesion (97) The Role of FAK in Muscle Development 48

50 The role of FAK during the major steps of muscle development has not been investigated as heavily as the integrin family but there is evidence that it has a major role for ensuring properly functioning tissue. In C2C12 cultures, it has been shown that FAK is activated during muscle differentiation (68). Clemente et al. (42) expanded upon this in their own C2C12 model. They found that phosphorylation of FAK Y397 was reduced approximately 50% immediately following differentiation compared to the proliferation state. This level then increased linearly until it was 2.5 fold higher than the proliferation state after 5 d. They also mutated Y397 and saw a decrease in proliferation markers and in increase in differentiation markers, although the cells themselves didn t differentiate. Furthermore, phosphorylated FAK Y397 was needed for myoblast fusion (42). In skeletal muscle myoblast cultures, different levels of certain integrin subunits can either activate or inhibit FAK signaling. Ectopically elevating the α5 subunit increased the FAK activation response while elevation of the α6 subunit decreased FAK activity and it appears to work through the β1a subunit (132). The decrease in FAK activity was seen concurrently with the lack of cell proliferation while the increase in activity was noted to correspond to myoblast proliferation. Adhesion of the α5β1 integrin to fibronectin increases FAK activation and leads to an increase in cell spreading in the presence of protein kinase C. This increase in FAK activation is decreased following inhibition of protein kinase C, demonstrating a potential regulatory marker during muscle development (52). In contrast, α7-null mice have an increased expression of activated FAK (131). Quach et al. (126) performed an elaborate study using an in vivo mouse model and an in vitro C2C12 culture model. The in vivo model was able to inhibit FAK in a satellite cell specific manner. This resulted in improper myotube formation following injury. In the in vitro model, FAK activation was increased at 8 and 18 h after the 49

51 addition of serum and was quickly preceded by increases in myogenin. FAK activity returned to baseline levels 48 h after serum addition. Inactivation of FAK resulted in an inhibition of myoblast fusion and this inhibition was coincided with a decrease in membrane protein expression, principally the β1d integrin and caveolin 3 (126). There is data suggesting that FAK is necessary for costamere genesis. Mouse myoblast cultures showed a gradual increase in cellular order upon formation of myofibrils. Introduction of FAK sirna led to a loss of mature costameres, an increase in nascent costameres and disruption to myofibrillar organization (127) FAK and the Response to Loading FAK is mainly incorporated to the integrin part of the costamere but it has been located in the dystroglycan complex in bovine synapses (38). Its principal function in skeletal muscle is with the integrin, however, and thus it is a prime candidate to relay the stretch signal from the integrins to the cytoplasm both from sarcolemma and from the MTJ (9). In mouse L6 muscle cultures, cyclic stretch increases FAK phosphorylation 15 min after stretch and stays elevated for 60 min (7). Flück et al. (56) were the first to describe FAK in developed muscle. They found that FAK was localized to focal adhesions in the sarcolemma, although this distribution was not regular. Total and active FAK expression increased during a 10% body weight loading of the rooster latissimus dorsi. Soleus overload by gastrocnemius ablation increased FAK expression 1 and 8 d post-ablation (56). Finally, they noted an increase in FAK following myotube formation (56). There are fiber type differences in FAK expression. The soleus has higher levels of baseline 50

52 FAK compared to the gastrocnemius or plantaris in rats (70). Paradoxically, 7 d of hindlimb unloading increased FAK expression in the soleus but decreased in fast-twitch muscle. FAK Y397 phosphorylation was severely decreased in the soleus of these rats (70). Lastly, all fiber types responded to overload at both 1 and 8 d post-gastrocnemius ablation and showed an increase in total FAK expression and FAK Y397, with the exception that the plantaris had no change in FAK Y397 1 d post-surgery (70). Interestingly, in human vastus lateralis that was unloaded by cast for 21 d, the phospho to total expression ratio of FAK was decreased from 0-10 d following unloading with no change occurring from d (50). A similar model by the same lab tried a nutritional supplementation to inhibit muscle atrophy during 14 d of unloading. They saw decreases in phosphorylated FAK Y576/577 in the immobilized leg compared to the control leg with no difference between those dosed with amino acids (67). Bedrest can lead to decreases in total FAK expression. During 34 d of bedrest, men had an approximately 20% decrease in FAK at days 8 and 34 (101). The role of the actual molecular changes with increased FAK expression was testing by using an electro-pulse transfer of FAK or its competitive inhibitor FAK-related non-kinase (FRNK). In this robust experiment, it was found that the overexpression of FAK led to increased mrna production of mitochondrial, metabolic, contractile, signaling and protease-based genes (54). Delivery of FRNK led to decreases in those gene categories listed. Overexpression of FAK led to large increases in myosin heavy chain 1 and changes in muscle fiber size. Interestingly, overexpression of the soleus with FAK and overloading caused an increase in myosin heavy chain 1, a decrease in myosin heavy chain 2, slower muscle contraction and relaxation and 51

53 double the tetanic force production (54). These changes, especially the changes in cellular signaling, were followed up by investigating whether electro-pulse-mediated FAK delivery has a role in the p70s6k response to load. It was found that p70s6k activation occurs roughly 24 h post- FAK overexpression and loading and this is approximately 18 h after peak of total and phosphorylated FAK (91). Intriguingly, there was no difference in Akt, a prominent protein upstream of p70s6k, activation or expression, providing evidence that this may be an Aktindependent relationship (91). Overexpression of FAK drives expression of other costameric proteins. Using the same model as the previous studies, vector-driven FAK increased the levels of β1 integrin and meta-vinculin (a scaffolding protein), although there were no changes in α7 or γ-vinculin (92). These same study also reinforced previous literature as in vivo stretch of the soleus increase FAK phosphorylation at 1 and 24 h post-stretch (92). Resistance training is another mode of muscle stress that has been thought to signal through FAK. Four sets of 10 repetitions at the 10-rep max of leg press and knee extensions did not have an affect on FAK, either total or phosphorylated, in young males (66). This is contrasted by data that suggest that trained and untrained males doing knee extensions or one-legged cycling have increased FAK Y576/577 activation immediately after exercise (164). Both resistance training and aerobic training led to an increased phospho to total ratio at baseline compared to pretraining (164). Nine-weeks of unilateral leg extension on a device that provided both eccentric and concentric loading increased FAK Y397 activation by 30% after 10 sessions and 190% after 24 sessions (101). Muscle loading via 12 wk of downhill skiing increased FAK concentration and this increase was seen in males, not females (58). 52

54 2.3.4 Neurological Input and FAK in Skeletal Muscle There is evidence that neurological input from the motor neuron may dictate FAK expression. Immunohistochemistry was utilized to locate FAK in soleus and extensor digitorum longus (EDL) mice. In slow-twitch muscle, 89% of muscle fibers had FAK localized to the sarcolemma while only 50% of muscle fibers had FAK at the sarcolemma in EDL muscle (57). Further, in the EDL, only 10% of the type IIB fibers had FAK at the sarcolemma. By cross-innervating the muscle, it was found that slow-twitch fibers were reduced by approximately 20% and FAK expression at the sarcolemma was reduced to 60% in the remaining type 1 fibers (57) FAK and the PI3K Pathway The interaction between FAK and the Src homology (SH) 2 domain, a very important domain shared by many Src family kinases, is quite intimate. The most relevant SH2 containing proteins for this discussion are csrc (discussed in section 2.4) and phosphatidylinositol-3 kinase (PI3K). Both pathways can initiate at FAK and can direct anti-apoptotic and pro-hypertrophy signaling, with csrc being more apparent in cardiac tissue and PI3K in skeletal muscle. The role of PI3K and FAK is based upon autophosphorylation of FAK. Following integrininitiated FAK Y397 phosphorylation, the SH2 domain of PI3K binds to FAK s kinase domain, increasing PI3K activity through phosphorylation of the p85 subunit (41). In a follow-up study 53

55 from the same lab, it was determined that FAK Y397 phosphorylation was absolutely necessary for this interaction (40). The PI3K pathway is a well-known and characterized pathway that responds to nutritional changes, mainly insulin and amino acids, growth factors (IGF-1) and changes in muscle loading (8; 17; 69). The pathway consists principally of PI3K, Akt, mtor and p70s6k. PI3K is composed of a 110 kda catalyzing subunit and an 85 kda regulatory subunit. PI3K relays its activation signal by phosphorylating phosphatidylinositide-4,5 (PIP2) to create PIP-3,4,5 (PIP3). PIP3 then activates PIP3-dependent kinase 1 (PDK1). PDK1 can then phosphorylate Akt at T308 (65). Akt activates mtor at S2448 and mtor reciprocally activates Akt at S473. Activated mtor is a major nexus for cell growth and protein synthesis. mtor relays the signal to p70s6k and forward to sarcomeric gene transcription and protein synthesis (65). While there is no direct evidence of FAK s role in protein synthesis, there are ways in which FAK can mediate the hypertrophic machinery of skeletal muscle. As mentioned above, vectordriven overexpression of FAK increases p70s6k phosphorylation and s6k activity (91) and cyclic strain in mouse muscle culture has shown a strong correlation between FAK activation and Akt kinase activity (7). FAK has been shown to interact with the IGF-1 receptor and insulin (4). 54

56 There is evidence that FAK can interact with proteins downstream of PI3K and Akt. Tuberous sclerosis complex (TSC) 1 and 2 are proteins that cause hamartomas, benign abnormal growths. TSC1 and TSC2 form a signaling complex and this complex is a potent upstream mediator of mtor activity. When unphosphorylated, TSC1/2 prohibits Rheb-induced activation of mtor (81). TSC1/2 is phosphorylated by Akt, thus inhibiting its affect on mtor and increasing mtor signaling. TSC1 has no determined enzymatic capability but TSC2 contains a Rheb binding site (63). FAK can phosphorylate TSC2 in vivo. In kidney cell cultures, FAK phosphorylates TSC2 in a csrc-independent manner and regulates phosphorylation of p70s6k and 4-eukaryotic binding protein-1 (4eBP1) (63). Furthermore, growth of skeletal muscle cell cultures is dependent upon FAK interacting with TSC2. Inhibition of FAK in cell culture shows decreased TSC2 phosphorylation and decreased signaling through p70s6k in the presence of IGF-1 (49). 2.4 csrc csrc is the founding member of Src family of protein kinases, a distinct but highly similar family consisting of nine proteins. Originally discovered as a cancer causing protein in chicken in 1911, it has been widely investigated in many cell types, with the exception of skeletal muscle. Similarly to FAK, csrc has been most heavily studied in cancer cells. csrc was the first protooncogene discovered in humans and plays a prominent role in cell proliferation, differentiation and cellular survival (130) Structure and Activation 55

57 The structure of csrc is preferentially in the inactive state, where its active site is buried within the protein because of intradomain binding. The N-terminal portion of csrc contains a membrane-localizing myristoyl group attached to an SH4 domain. This is followed by a unique domain (what most differentiates it from other Src family kinases), an SH3 domain and an SH2 domain. The SH2 domain is connected to the protein kinase domain by a linker domain. The kinase domain contains the principal activation site of csrc, Y416. The C-terminal domain, or tail domain, contains the major inhibitory site of csrc, Y527 (130). When phosphorylated, Y527 binds to the SH2 domain and this binding is inhibited when Y527 is mutated to F527 (93). Harrison (76) adeptly describes the activation of csrc as a three part process: unlatching, unclamping, and switching.. Unlatching occurs when Y527 is dephosphorylated and the tail dissociates from the SH2 complex. Unclamping refers to a dissociation between the SH2 and SH3 domain interaction. When these domains separate it allows the kinase domain containing Y416 to open itself up for phosphorylation and allows the SH2 and SH3 domains to bind to substrates. The switch refers to the complete switch of activity from inactive to active (76). This transition from inactive to active state of csrc allows it to bind its SH2 domain to FAK following phosphorylation of FAK Y397 (135; 147). SH2 binding allows for csrc-induced phosphorylation of FAK Y576 and Y577 resulting in a fully activated FAK (30). This interaction creates a very strong and stable binding complex (43) csrc in Striated Tissue 56

58 The data for csrc in skeletal muscle is incredibly sparse. csrc has been shown to be susceptible to changes in stretch in muscle cultures but this is most likely through the integrin complex (175). Addition of PP2, a potent SFK inhibitor, to these stretched cells decreased csrc expression and cell proliferation (175). csrc colocalizes with the androgen receptor and triggers hypertrophic signaling of the Akt pathway (143). Our lab has shown that csrc, both total and phosphorylated Y416, is not effected by eccentric exercise or heat shock (manuscript in review). In cardiac tissue, this pathway seems to be more responsive to stretch. In cat ventricles overloaded by banding, csrc increased expression, both total and activated, and became localized to the cytoskeletal architecture within 4 h of banding, reached maximal levels at 48 h, and then returned to baseline 7 d post-banding (96). csrc kinase activity of the myocardium increased 3- fold within 10 d of aortic banding in guinea pig. These levels stayed elevated for 56 d (145). Passive stretching of neonatal rat ventricular myocytes increased csrc activation and this was blunted with the addition of PP2, a potent inhibitor of csrc activation (150). csrc has the potential to create cellular hypertrophy through extracellular signal-related kinase 1/2 (ERK1/2). ERK1/2 is a member of the mitogen activated protein kinase (MAPK) family. The relationship between csrc and ERK1/2 has not been well-established in skeletal muscle but in pulmonary epithelial cells, strain-induced ERK1/2 activation is ablated following inhibition of both FAK and csrc (39). The expression of ERK1/2 is easily manipulated through exercise. Eccentric contractions have been shown to activate ERK1/2 by different mechanisms compared to concentric contractions (167). Resistance training (165), marathon running (173), eccentric 57

59 exercise (20) and static stretch (82) have all been shown to increase ERK1/2 activity. ERK1/2 can translocate to the nucleus and interact with initiation factors (34) or it can remain in the cytosol and relay activation signals (166). For hypertrophic signaling, ERK1/2 is necessary for maintenance of cultured muscle as pharmaceutical inhibition created atrophied and unhealthy cells (140). ERK1/2 can phosphorylate mtor. Decreased ERK1/2 function decreases mtor activity in an Akt-independent manner, suggesting that ERK1/2 directs its own parallel pathway following administration of lipoic acid or insulin (166). 2.5 Acetaminophen Acetaminophen (APAP) is a widely consumed over-the-counter drug used to treat pain and muscle soreness. APAP is a non-specific cyclooxygenase (COX) inhibitor. Arachidonic acid is created from phospholipids by membrane-bound lipases. COX then converts arachidonic acid into precursor prostaglandins. Following creation of these precursor prostaglandins, specific enzymes further process them into specific isoforms. Prostaglandins are then able to function as autocrine or paracrine molecules (154) APAP and Skeletal Muscle In skeletal muscle, the major regulatory prostaglandins for hypertrophy and atrophy are prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2), respectively (83; 110). Both of these prostaglandins are increased following an acute eccentric exercise protocol but ingestion of APAP and ibuprofen attenuated this response (153). Non-prescription levels of APAP and 58

60 ibuprofen supplementation have been demonstrated to blunt post-exercise protein synthesis gains following an eccentric exercise protocol as measured by fractional synthetic rate (156). The mechanism behind this is most likely changes in the COX-1 or COX-2 variants, and not isoforms containing an extra intron from COX-1, in skeletal muscle as COX-1 was constitutively active and COX-2 readily changed 4 and 24 h post-eccentric exercise when analyzed by RT-PCR (162). Although COX-2 mrna was significantly higher there was not a concomitant change in COX-2 protein expression (162). To further elucidate these results, male subjects were given celecoxib, a COX-2 specific inhibitor. The men then completed an eccentric exercise bout and had their FSR measured along with other markers of COX-2. Those that took the COX-2 inhibitor had the same post-exercise gain in protein synthesis as well as gains in COX-2 mrna (23). This provided evidence that COX-2 is most likely not the major effector of APAP-induced decreases in protein synthesis even though COX-1 protein expression remained unchanged and mrna increased following exercise (23). COX-1 and COX-2 activity were increased 4 h post-exercise with COX-2 activity remaining elevated for 24 h (36). This change in COX-1 activity was not correlated with an increase in protein expression but COX-2 protein expression was elevated 24 h post-exercise (36). Two mechanisms behind this COX-induced change in muscle have been proposed based upon mrna data between COX-inhibitor and placebo-controlled subjects. One of these is an increase in the receptor for PGF2α which would increase sarcolemmal sensitivity (155). The other mechanism is a decreased amount of PGE2, leading to decreases in atrophic signaling caused by interleukin-6 (IL-6) and muscle RING finger protein-1 (MuRF1) (155). 59

61 The role of APAP and chronic exercise has a paradoxical effect on muscle protein synthesis. Elderly men and women completed 12 wk of leg extension exercises at 3 sets of 10 repetitions. Weight was increased every two weeks to ensure proper training progress. Subjects in the APAP group took the maximal over-the-counter dose (4,000 mg) every day throughout the training program. The APAP group had larger gains in muscle size and strength compared to a placebo group. All groups increased COX-1 protein expression and COX-1 and -2 mrna (152) APAP and Tendon Function Tendons, similarly to skeletal muscle, respond to chronic loading through hypertrophy and gains in function (139). However, APAP supplementation and function in tendon does not appear to respond as skeletal muscle does. Following APAP supplementation in elderly males and females for a 12 wk resistance training protocol, mean tendon size increased but strain, stiffness and elastic modulus were negatively affected at both peak and common forces (35). Decreases of this type could lead to losses in tendon function and performance (139). One potential mechanism for this loss in performance is a decrease in collagen crosslinking. In rats that were given daily doses of APAP or saline and exercised or remained sedentary, those that were given APAP had lower levels of hydroxylyslpyridinoline, a molecule necessary for crosslinking collagen (37) 2.6 Conclusion The α7β1 integrin and related focal adhesion respond to various muscle stresses (Fig. 7). They are necessary for both proper muscle development and proper muscle function. Various cellular 60

62 pathways are regulated by these membrane proteins but in skeletal muscle it appears that, ultimately, they transmit force, anchor and protect the sarcolemma and signal for the cell to grow and become healthy. Exercise is a prominent way to activate these proteins, with signals relaying mainly through FAK. FAK activates multiple proteins in a scenario where there may be three effected pathways: PI3K/AKT, TSC2/mTOR or csrc/erk1/2. Increases in the expression of these proteins result in protein synthesis and hypertrophy while decreases result in protein degradation and atrophy. APAP affects skeletal muscle through alterations in PGF2α and PGE2 by COX enzymes. APAP is a non-specific COX inhibitor and while APAP supplementation blunts the acute protein synthesis response following exercise, chronic exercise leads to greater muscle size and strength gains. These changes may be related to increases in receptor sensitivity and decreases in atrophic markers. The gains in muscle function are coupled with decreases in tendon function. APAP decreases tendon stiffness and modulus while increasing size and strain. Losses in collagen crosslinking have also been seen. The above reflect the role of exercise and APAP supplementation upon skeletal muscle health. Any change in skeletal muscle size and function may have role within the focal adhesion since it is a nexus of force and stretch signaling. The following studies will help unravel the role of the 61

63 focal adhesion within skeletal muscle following distinct interventions. Figure 7. Signaling capabilities of the α7β1 integrin. Most signals get relayed through FAK. FAK can directly activate potential hypertrophic pathways via PI3K and csrc. FAK can inhibit the activation of TSC2, thus inhibiting an inhibitor, another potential hypertrophic manner. ILK bound to the α subunit has been shown to directly activate Akt. Abbreviations: ILK-integrin-linked kinase; FAK-focal adhesion kinase; PI3K-phosphatidylinositol-3 kinase; mtor-mechanistic target of rapamycin; ERK1/2-62

64 Chapter 3: Methods 63

65 3.1 IASTM and the α7β1 Integrin Pathway Subjects Eleven healthy males (age: 23 ± 3 years; weight: 83 ± 11 kg; height: 181 ± 7 cm) volunteered to participate in this study. They were screened for neurological disorders, musculoskeletal injuries and general contraindications to exercise in accordance to the American College of Sports Medicine guidelines. All volunteers filled out an informed consent and health history questionnaire. The investigation was approved by the Institutional Review Board for Human Subjects at the University of Kansas and followed the ethical guidelines for treatment of human participants outlined in the Declaration of Helsinki. IASTM IASTM (Graston Technique, Indianapolis, IN) was performed by a practicing certified athletic trainer trained to perform the technique on the lower limbs. The treatment was isolated to the planter flexors, which were divided into 4 treatment areas. Each section received 2 sets of 7 strokes in both proximal and distal directions with a convex and concave instruments. The instruments are made of stainless steel and designed specifically for IASTM (Graston Technique, Indianapolis, IN). To ensure that the correct angle was maintained throughout the procedure, a bubble level was applied to both instruments at 45 to provide the clinician with a consistent visual reference. ELF pressure sensors (Tekscan, Boston, MA) were placed on the contact surface to ensure consistent clinical pressure. The IASTM lasted a total of 7-9 min. 64

66 All participants came into the lab 4 times. They were instructed to refrain from exercise 24 h prior to the first laboratory visit as well as refraining from exercise during the subsequent days of data collection. On the first day the participants came to the lab (0h), they received a muscle biopsy from the gastrocnemius (GST) on the control leg (CL), which received no IASTM. Immediately afterwards, participants received IASTM on the treatment leg (TL). Participants returned to the lab and had muscle biopsies taken from the TL at 24 hours (24h), 48 hours (48h) and 72 hours (72h) post-iastm. Biopsy All participants received percutaneous muscle biopsies (~100 mg) from the lateral portion of the GST of the CL at 0h and from the TL at 24h, 48h and 72h. A topical antiseptic (Betadine) was applied to the biopsy site. This was followed by an injection of 3 cc of 2% lidocaine. There was a 5 min wait to ensure the area was sufficiently anesthetized then an incision approximately 0.5 cm wide and one cm deep was made using a #11 scalpel. For each biopsy of the TL, the incision site was moved 1 cm distally. All samples were immediately cut into ~20 mg pieces and placed in liquid nitrogen for storage. Protein Quantification The muscle was homogenized with a 10:1 volume to weight ratio in tissue extraction buffer (T- PER; Thermo-Scientific, Rockford, IL) with the addition of protease and phosphatase inhibitors 65

67 (Halt 100x Protease and Phosphatase; Thermo-Scientific, Rockford, IL) and phenylmethanesulphonylfluoride (PMSF; Nunc; Nalgene International, Rochester, NY) using a glass on glass mortar and pestle. The homogenate was kept on ice, vortexed every 10 min for 30 min, then centrifuged at 3000 rpm for 5 min. Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Pierce BCA Protein Assay Kit; Pierce, Rockford, IL). SDS-PAGE and Western Immunoblotting Electrophoresis was performed via SDS-PAGE (Mini-PROTEAN 3 cell and PowerPac High- Current Power Supply; Bio-rad, Hercules, CA). 80 µg of protein were mixed with HES buffer (20 mm HEPES, ph 7.4, 1 mm EDTA, and 250 mm sucrose) and a 5x lane marker and lysing buffer (Fisher Scientific, Waltham, MA) then loaded into poly-acrylamide gels. Gels were ran at a constant 0.07 amps in 1x running buffer (25 mm Tris, 192 mm glycine and 0.1% SDS) for min. Proteins were transferred onto a PVDF membrane (Amershand Hybond; GE Lifesciences, Piscataway, NJ) following activation in methanol. The proteins were transferred at 0.20 amps for 120 min in transfer buffer (25 mm Tris, 192 mm glycine and 10% methanol) and then blocked in 5% dry milk/tris-buffered saline with Tween 20 (TBS-T; ph 7.6) solution for 1 h. After blocking, membranes were incubated overnight at 4 C in primary antibody (alpha7 integrin and beta1 integrin; Santa Cruz Biotechnology, Santa Cruz, CA; FAK, FAK Y397, csrc, csrcy416, ERK1/2 and ERK1/2 T/202Y/204, and alpha-tubulin; Cell Signaling, Beverly, MA) diluted in a 66

68 1% milk/tbst solution. Primary antibody dilutions were 1:250 for α7 integrin, 1:500 for phosphorylated and total csrc and 1:1000 for the rest (µl antibody:µl 1% milk solution). The following day membranes were rinsed in 3x10 min washes in TBS-T and then placed with an anti-rabbit horseradish peroxidase linked (HRP) secondary antibody (Anti-rabbit and anti-mouse IgG; Cell Signaling, Beverly, MA) with a 1:1000 solution for the α7 integrin and total and phosphorylated csrc and 1:2000 dilution for all other proteins for an hour. Membranes were washed again with a 3x10 min set with TBS-T then incubated with a horseradish peroxidase chemilumenescent (Amersham ECL Western Blotting System; GE Lifesciences, Piscataway, NJ) for 5 min and then developed with a protein imaging system (Fluorchem HD2 with an upgraded 16 bit 4.2 megapixel camera with a F/0.95 lens; ProteinSimple, Santa Clara, CA). All membranes analyzed for proteins with active phosphorylation sites (FAK, csrc and ERK1/2) were stripped using a mild stripping buffer, confirmed for absence of a chemilumenescent signal, then reprobed for the phosphorylation site of interest. Densitometry software (Fluorchem HD2; ProteinSimple, Santa Clara, CA) was used to quantify pixel brightness. Each sample was quantified three times and averaged. Samples were normalized to a control sample that was loaded onto each gel to limit between gel differences. Samples were also normalized to the loading control (alpha-tubulin). Statistics 67

69 All statistics were carried out using SPSS 20.0 (IBM, Armonk, New York). Separate 1x4 one-way repeated measures ANOVAs were completed for each protein. Significance was set at <0.05. Data are presented as ± standard deviation (SD). 3.2 APAP Supplementation and 8 wk of Aerobic Exercise All methods related to animal handling, training and muscle excision were the same as reported by Carroll et al. (37). These methods are briefly summarized below. Animals Eight week-old male Wistar rats (n=24) were obtained from Charles River Labs (Wilmington, MA). Animals were familiarized with treadmill exercise with a 2-wk acclimation period and assigned to one of four treatment groups (n=6 per group): sedentary+placebo (SED+PLAC), sedentary+apap (SED+APAP), exercise+placebo (EX+PLAC) or exercise+apap (EX+APAP). Rats were caged in pairs, were given food and water ad libitum, and maintained on a 12 h lightdark cycle. The study was approved by the Midwestern University Institutional Animal Care and Use Committee, and all animals were cared for in accordance to the recommendations in the Guide for the Care and Use of Laboratory Animals. Following completion of the 8 wk protocol, animals were euthanized and the soleus and gastrocnemius were extracted and immediately frozen in liquid nitrogen and stored at -80 C. Exercise Protocol 68

70 All rats completed a progressive treadmill exercise program lasting 8 wk. Rats were exercised 5 d/wk progressing to 60 min/d. Each session included 5 min of both a warm-up and cool-down. Speed and elevation were progressed to 20 m/min and 8 grade, respectively. Heart total protein content and total fat pad weight were measured as markers of exercise training effectiveness. Acetaminophen Administration Liquid APAP (100 mg/ml; Cypress Pharmaceutical, Madison, MS) was administered once daily via oral gavage (200 mg/kg). The amount of drug administered was sufficient to maintain therapeutic plasma levels for several hours each day without becoming toxic. The dose given was higher than the typical relative amount consumed daily by humans but this amount was required to equal the spinal APAP concentration following a typical dose given to humans. Control animals receiving the placebo were administered saline via oral gavage of equivalent volume ( ml). Animals were weighed weekly for adjustment of APAP dosing. Protein Quantification The soleus and gastrocnemius were homogenized in a glass on glass mortar and pestle with a cocktail of cellular extraction buffer (T-PER, ThermoScientific, Illinois, USA), phosphatase and protease inhibitors (ThermoScientific, Illinois, USA) and PMSF (Nunc: Nalgene International, New York, USA). Total protein concentration was determined by adding 1 µl of homogenized sample into 500 µl of deionized water and placed into a BCA assay (Pierce Scientific, Illinois, USA). Volume corresponding to 60 µg of sample protein was mixed with Laemlli sample buffer with DTT in a 1:2 ratio. Analysis of protein was carried out by SDS-PAGE and western immunoblotting. 69

71 SDS-PAGE Electrophoresis was performed via SDS-PAGE (Mini-PROTEAN 3 cell and PowerPac High- Current Power Supply; Bio-rad, Hercules, CA). Equal amounts of protein (60µg) were loaded into poly-acrylamide gels (6%, 10% and 12%). Gels were ran at a constant.05 amps in 1x running buffer for minutes. Western Immunoblotting Proteins were transferred onto a PVDF membrane (Amershand Hybond; GE Healthcare, Buckinghamshire, England) that had been activated in a 30 s methanol immersion. The proteins were transferred at 0.20 amps for 110 min in transfer buffer, Ponceau-stained to ensure equal protein loading, then blocked immediately after destaining in a 5% dry milk/tbs-t (ph 7.6) solution for 1 hr. After blocking, the membranes were placed in a primary antibody (α7 and β1 integrin; Santa Cruz Biotechnology, Santa Cruz, CA; FAK, FAK Y397, csrc, csrcy416, ERK1/2 and ERK1/2 T/202Y/204, p70s6k, p70s6k T389, 4eBP1 and 4eBP1 T70; Cell Signaling, Beverly, MA) diluted in a 1% milk/ TBS-T solution. Primary antibody dilutions were 1:250 for the α7 integrin, 1:500 for phosphorylated and total csrc and 1:1000 for the rest. Membranes were incubated overnight in a 4 C refrigerator. After incubation, membranes were rinsed in 3x10 min washes in TBS-T and then placed with an anti-rabbit horseradish HRP secondary antibody (Antirabbit and anti-mouse IgG; Cell Signaling, Beverly, MA) with a 1:1000 dilution for α7 integrin and 1:2000 dilution for the rest, for an hour. Following incubation, membranes were washed with a 3x10 min set with TBS-T. Membranes were then incubated with a horseradish peroxidase chemilumenescent (Amersham ECL Western Blotting System; GE Healthcare, 70

72 Buckinghamshire, England) for 5 min and then developed with a protein imaging system (Fluorchem HD2; ProteinSimple, Santa Clara, CA) Quantification Densitometry software (Fluorchem HD2; ProteinSimple, Santa Clara, CA) was used to quantify protein expression. Each sample was quantified three times and averaged. Samples were normalized to tricep control samples that were loaded on each gel. Statistics All statistics were carried out using SPSS 20.0 (IBM, Armonk, New York). Interactions for exercise and drug or main effects were investigated via one-way ANOVA. Significance was set at p<0.05. Main effects were analyzed with independent samples t-tests and a Bonferroni adjustment to control type 1 error. Data are presented in arbitrary units (AU) and as ± SD. 3.3 SCI and the Focal Adhesion The methods dealing with these animals have been previously reported (168). These methods are briefly described below. Animals Twenty male Wistar rats were used during this study each weighing approximately 250 g. Animals were house in a facility that was temperature and humidity controlled and had 12 h day:light cycle. Animals were given access to water and chow ad libitum. 71

73 Spinal Cord Injury Ten rats were exposed to spinal cord injury via complete T4 transection (SCI) or sham injury (SHAM) in which the spinous process was removed. Rats were left to their cage and monitored for 56 d post-surgery. At day 56, the gastrocnemius was excised after the rats were anesthetized via inhalation of 3-5% isoflurane. Animals were sacrificed via aortic transection. Protein Quantification The soleus and gastrocnemius were homogenized in a glass on glass mortar and pestle with a cocktail of cellular extraction buffer (T-PER, ThermoScientific, Illinois, USA), phosphatase and protease inhibitors (ThermoScientific, Illinois, USA) and PMSF (Nunc: Nalgene International, New York, USA). Total protein concentration was determined by adding 1 µl of homogenized sample into 500 µl of deionized water and placed into a BCA assay (Pierce Scientific, Illinois, USA). Volume corresponding to 80 µg of sample protein was mixed with Laemlli sample buffer with DTT in a 1:2 ratio. Analysis of protein was carried out by SDS-PAGE and western immunoblotting. SDS-PAGE Electrophoresis was performed via SDS-PAGE (Mini-PROTEAN 3 cell and PowerPac High- Current Power Supply; Bio-rad, Hercules, CA). Equal amounts of protein (60µg) were loaded 72

74 into poly-acrylamide gels (6%, 10% and 12%). Gels were ran at a constant 130 V in 1x running buffer for minutes. Western Immunoblotting Proteins were transferred onto a PVDF membrane (Amershand Hybond; GE Healthcare, Buckinghamshire, England) that had been activated in a 30 s methanol immersion. The proteins were transferred at 0.20 amps for 110 min in transfer buffer, Ponceau-stained to ensure equal protein loading and the blocked immediately after in a 5% dry milk/tbs-t (ph 7.6) solution for an hour. After blocking, the membranes were placed in a primary antibody (α7 and β1 integrin; Santa Cruz Biotechnology, Santa Cruz, CA; FAK, FAK Y397, csrc, csrcy416, ERK1/2 and ERK1/2 T/202Y/204, p70s6k, p70s6k T389, FAK, pfak 397 csrc, pcsrc 416, ERK1/2, perk1/2 202,204, p70s6k and pp70s6k 389 ; Cell Signaling, Beverly, MA) diluted in a 1% milk/ TBST solution. Primary antibody dilutions were 1:250 for α7 integrin, 1:500 for phosphorylated and total csrc and 1:1000 for the rest (µl antibody:µl 1% milk solution). Membranes were incubated overnight in a 4 C refrigerator. After incubation, membranes were rinsed in 3x10 min washes in TBS-T and then placed with an HRP-linked secondary antibody (Anti-rabbit and antimouse IgG; Cell Signaling, Beverly, MA) with a 1:1000 dilution for α7 integrin and 1:2000 for the rest, dilution for an hour. Following incubation, membranes were washed with a 3x10 min set with TBS-T. Membranes were then incubated with a horseradish peroxidase chemilumenescent (Amersham ECL Western Blotting System; GE Healthcare, Buckinghamshire, England) for 5 minutes and then developed with a protein imaging system (Fluorchem HD2; ProteinSimple, Santa Clara, CA 73

75 Quantification Densitometry software (Fluorchem HD2; ProteinSimple, Santa Clara, CA) was used to quantify pixel brightness. Each sample was quantified three times and averaged. Samples were normalized to control samples that were loaded on each gel. Statistics All statistics were carried out using SPSS 20.0 (IBM, Armonk, New York). Independent-samples t-tests were used to determine differences in the means. Significance was set at p<0.05 with a Bonferroni adjustment to limit type 1 error. Data are presented in AU and as ± SD. 74

76 Chapter 4: Results 75

77 4.1 IASTM and the α7β1 Integrin Pathway There was no effect for time on either the 120 kda α7 subunit (p=0.409) or the 70 kda variant (p=0.590) (Fig. 1). There was no difference in expression of the β1 subunit (p=0.890) (Fig. 2) or total or phosphorylated FAK (p=0.771 and p=0.409, respectively) (Fig 3). There were no differences between total or phosphorylated csrc (p=0.183 and p=0.091, respectively; Fig. 4A and 4B) or ERK1/2 (p=0.429 and p=0.141; Fig. 4C and 4D). Furthermore, there were no differences for any protein at any timepoints following conservative pairwise comparisons. Fig. 1 Representative blots of expression of the alpha7beta1 integrin. There was no change over the course of 72 h in either the A) 120 kda subunit B) the 70 kda subunit of the alpha7 integrin. Data are presented ± SD. 76

78 Fig. 2 The expression of the beta1 subunit remains unchanged at 24 h, 48 h and 72 h post- IASTM. Shown with representative blots. Data are presented ± SD. Fig. 3 The response of FAK to IASTM with an accompanying representative blot. A) Total FAK expression is constant over the course of data collection. Furthermore, B) phosphorylation of the main active site of FAK also remains constant. Data are presented ± SD. 77

79 Fig. 4 csrc and ERK1/2 are not responders to IASTM at 24 h, 48 h and 72 h post-iastm. A) Total csrc remains unchanged as well as B) phosphorylated csrc. Representative images of C) Total ERK1/2 and D) phosphorylated ERK1/2 are not effected by IASTM. Data are presented ± SD. 4.2 APAP Supplementation and 8 wk of Aerobic Exercise Soleus Interaction Effects There were no significant interaction effects between the 120 kda α7 integrin (p=0.338; Fig. 5A), the 70 kda α7 integrin (p=0.447; Fig. 5B) or the β1 integrin (p=0.989; Fig. 5C). FAK showed no interaction effect for either total or phosphorylated Y397 (p=0.054 and p=0.150, respectively; Fig. 6A and 6B). There were no interaction effects in total or phosphorylated csrc (p=0.126 and p=0.685, respectively; Fig. 6C and 6D), ERK1/2 (p=0.998 and p=0.202, 78

80 respectively; Fig. 7A and 7B), p70s6k (p=0.516 and p=0.515, respectively; Fig. 8A and 8B) or 4EBP1 (p=0.092 and p=0.770, respectively; Fig.8C and 8D). Fig. 5 Interaction effects for the A) 120 and B) 70 kda α7 integrin and the C) β1 integrin in the soleus. There were no significant interactions between any of the proteins. Data are presented ± SD. 79

81 Fig. 6 There were no significant interaction effects for A) total FAK and B) phosphorylated FAK or C) total csrc and D) phosphorylated csrc in rat soleus. Data are presented ± SD. Fig. 7 80

82 Graphs of A) total ERK1/2 and B) phosphorylated ERK1/2 in the rat soleus. There were no significant interaction effects. Data are presented ± SD. Fig. 8 Representative graphs of the hypertrophic proteins p70s6k and 4eBP1 in the soleus. There were no significant interaction effects for A) total p70s6k and B) phosphorylated p70s6k or C) total 4eBP1 and D) phosphorylated 4eBP1. Data are presented ± SD. APAP Main Effect There were differences between the main effect for APAP supplementation for multiple proteins compared to a Bonferroni-corrected α number of p= Rats that consumed APAP had higher levels of the 70 kda α7 integrin (p<0.001; Fig. 9B), total and phosphorylated FAK, (p<0.001 and 81

83 p=0.001, respectively; Fig. 10A), total and phosphorylated csrc (p<0.001 for both; Fig. 10B) and total p70s6k (p<0.001; Fig. 11A). There were no significant differences for the 120 kda α7 integrin (p=0.679; Fig. 9A), β1 integrin (p=0.806; Fig. 9C), phosphorylated p70s6k (p=0.014; Fig. 11A), total or phosphorylated 4eBP1 (p=0.008 and p=0.014, respectively; Fig. 11C and 11D) and total or phosphorylated ERK1/2 (p=0.687 and p=0.406, respectively; Fig. 12). Fig. 9 Expression of the integrin subunits when collapsed for APAP supplementation in the soleus. A) the 120 kda α7 subunit had no significant changes. B) Significant changes in the 70 kda α7 integrin and C) no change in expression of the β1 subunit. D) Representative blots shown in comparison to the 43 kda Ponceau stain. (*) denotes increased expression (p<0.004) compared to PLAC. Data are presented ± SD. 82

84 Fig. 10 Main effect representation of APAP supplementation in the soleus. There were significant increases in A) total FAK and B) phosphorylated FAK as well as increases in C) total csrc and D) phosphorylated csrc. E) Representative blots shown in comparison to the 43 kda Ponceau stain. (*) denotes increased expression (p<0.004) compared to PLAC. Data are presented ± SD. 83

85 Fig. 11 Increases in hypertrophy-related proteins following APAP supplementation in the soleus. A) total p70s6k, B) phosphorylated p70s6k, C) total 4eBP1 and D) phosphorylated 4eBP1. (*) denotes increased expression (p<0.004) compared to PLAC. Representative blots shown in comparison to the 43 kda Ponceau stain. Data are presented ± SD. 84

86 Fig. 12 No change was seen with APAP supplementation in the soleus with A) total or B) phosphorylated ERK1/2. C) Representative blots shown in comparison to the 43 kda Ponceau stain. Data are presented ± SD. Exercise Main Effect Exercise had no effect on the expression of any protein investigated in the soleus. There were no differences for the 120 kda α7 integrin (p=0.890; Fig. 13A) or 70 kda α7 integrin (p=0.159; Fig. 13B), β1 integrin (p=0.107; Fig. 13C) total or phosphorylated FAK (p=0.181 and p=0.051, respectively; Fig. 14A and 14B), total or phosphorylated csrc (p=0.753 and p=0.312, respectively; Fig. 14C and 14D), total or phosphorylated p70s6k (p=0.390 and p=0.520, respectively; Fig. 15A and 15B) and total or phosphorylated 4eBP1 (p=0.393 and p=0.340, respectively; Fig. 15C and 15D). total or phosphorylated ERK1/2 (p=0.209 and p=0.445, respectively; Fig. 16). 85

87 Fig. 13 Aerobic exercise training does not increase the expression of either the α7 or β1 integrin subunits in the soleus. A) Expression of the 120 kda B) the 70 kda α7 integrin and C) β1 integrin. Data are presented ± SD. 86

88 Fig. 14 No alterations in focal adhesion signaling following exercise training in the soleus. A) Eight wk of exercise does not induce changes in total FAK expression or B) levels of FAK activation. There were no changes seen in C) total csrc or D) phosphorylated csrc expression following aerobic exercise training. Data are presented ± SD. 87

89 Fig. 15 There were no changes in the expression of principal hypertrophy proteins following exercise training in the soleus. Expression of both A) total and B) phosphorylated p70s6k and C) total and D) phosphorylated 4eBP1 remained unchanged. Data are presented ± SD. Fig. 16 In the soleus, A) total and B) phosphorylated ERK1/2 remained unchanged following 8 wk of aerobic exercise training. Data are presented ± SD. 88

90 4.2.2 Gastrocnemius Interaction Effects There were no significant interactions for APAP supplementation and exercise in the rat gastrocnemius for the α7 120 kda integrin subunit (p=0.351; Fig. 17A). There was no detection of the 70 kda subunit. There were no interaction effects for the β1 integrin (p=0.671; Fig. 17B), total or phosphorylated FAK (p=0.124 and p=0.119, respectively; Fig. 18A and 18B), total or phosphorylated csrc (p=0.658 and p=0.135, respectively; Fig. 18C and 18D), total or phosphorylated ERK1/2 (p=0.128 and p=0.458, respectively; Fig. 19A and 19B), total or phosphorylated p70s6k (p=0.516 and p=0.719, respectively; Fig. 20A and 20B) and total or phosphorylated 4eBP1 (p=0.108 and p=0.130, respectively; Fig. 20C and 20D). Fig. 17 In the gastrocnemius, there were no APAP and exercise interactions for the A) 120 kda α7 or B) β1 integrin subunits. The 70 kda α7 integrin subunit was not detected. Data are presented ± SD. 89

91 Fig. 18 There were no interaction effects for APAP supplementation and aerobic exercise training in the gastrocnemius for proteins of the focal adhesion. A) total and B) phosphorylated FAK remained unchanged as well as C) total and D) phosphorylated csrc. Data are presented ± SD. Fig. 19 Exercise training and APAP administration does not interact to induce changes in the gastrocnemius for ERK1/2. Shown are representative graphs for expression of A) total and B) phosphorylated ERK1/2. Data are presented ± SD. 90

92 Fig. 20 Hypertrophic signaling proteins in the gastrocnemius show no evidence of an APAP and exercise training interaction. A) total and B) activated p70s6k showed no change along with C) total and D) phosphorylated 4eBP1. Data are presented ± SD. APAP Main Effect There was a significant decrease in the main effect for APAP supplementation for phosphorylated p70s6k (p<0.001; Fig. 24B). While not significant, there was a large mean decrease in phosphorylated FAK (p=0.007; Fig. 22B). There were no differences in the α7 integrin (p=0.825; Fig. 21A), β1 integrin subunit (p=0.978; Fig. 21B), total FAK (p=0.487; Fig. 22A), total or phosphorylated csrc (p=0.872 and p=0.742, 91

93 respectively; Fig. 22C and 22D), total or phosphorylated ERK1/2 (p=0.242 and p=0.505, respectively; Fig. 23), total p70s6k (p=0.187; Fig. 24A), or total or phosphorylated 4eBP1 (p=0.295 and p=0.783, respectively; Fig. 24C and 24D). Fig. 21 When collapsed into the main effect of APAP supplementation for the gastrocnemius, there were no significant decreases in either the A) 120 kda α7 integrin or B) the β1 subunit. C) Representative blots shown in relation to the 43 kda Ponceau stain line. Data are presented ± SD. 92

94 Fig. 22 APAP supplementation decreases activation of the focal adhesion in the gastrocnemius. A) There was no change in expression of total FAK based upon APAP administration. B) Significant decreases in FAK phosphorylation in rats administered APAP. There were no significant changes in C) total and D) phosphorylated csrc. E) Representative Western blots shown in relation the 43 kda Ponceau stain. Data are presented ± SD. 93

95 Fig. 23 Investigation of an APAP-induced change in ERK1/2 shown no significant changes in either A) total expression or B) phosphorylated expression in the rat gastrocnemius. C) Representative immunoblots compared to the 43 kda Ponceau stain. Data are presented ± SD. 94

96 Fig. 24 There were changes in the hypertrophic signaling expression in APAP-administered rat gastrocnemius. A) Total expression of p70s6k remained unchanged. B) There was a significant decrease in expression of p70s6k phosphorylation following eight wk of APAP supplementation. There was no significant change in C) total or D) phosphorylated 4eBP1. E) Representative Western immunoblots for the mentioned proteins. The 43 kda Ponceau stain demonstrates equal protein loading. ( ) denotes increased expression (p<0.004) compared to APAP. Data are presented ± SD. 95

97 Exercise Main Effect There were no significant differences for exercise. However there were large mean changes indicating an increase in phosphorylated FAK (p=0.049; Fig. 26B) and phosphorylated ERK1/2 (p=0.028; Fig. 28B). There was no change in the α7 integrin, (p=0.351; Fig. 25A), β1 integrin (p=0.816; Fig. 25B), total FAK (p=0.451; Fig. 26A), total or phosphorylated csrc (p=0.282 and p=0.673, respectively; Fig. 26C and 26D), total or phosphorylated p70s6k (p=0.390 and p=0.320, respectively; Fig. 27A and 27B), total or phosphorylated 4eBP1 (p=0.336 and p=0.125, respectively; Fig. 27C and 27D) and total ERK1/2 (p=0.507; Fig. 28A). Fig. 25 Exercise training does not affect integrin expression in rat gastrocnemius muscle. There were no significant differences in the expression of the A) 120 kda α7 or B) β1 integrin following eight wk of aerobic exercise. Data are presented ± SD. 96

98 Fig. 26 The focal adhesion proteins FAK and csrc remain unchanged following uphill aerobic exercise training in the gastrocnemius. There were no significant changes in A) total or B) phosphorylated FAK as well as no changes in A) total or B) phosphorylated csrc. Data are presented ± SD. 97

99 Fig. 27 Aerobic exercise training induced no change in hypertrophic signaling proteins in the rat gastrocnemius. There was no change in expression of A) total or B) activated p70s6k. Further, the downstream protein 4eBP1 showed no changes in expression of its C) total or B) phosphorylated form. Data are presented ± SD. Fig. 28 Following eight wk of aerobic exercise A) total ERK1/2 remained unchanged. B) Exercise induced a doubling, although not-significant, in ERK1/2 activation. Data are presented ± SD. 98

100 4.3 SCI and the Focal Adhesion There were no significant differences for either integrin subunit. Interestingly, there was a large mean increase in the SCI group in expression of the 120 kda α7 integrin (p=0.026; Fig. 29) but no similar change in the β1 integrin (p=0.240; Fig. 30A), The SCI group had significantly lower levels of total (Fig. 30B) and phosphorylated (Fig. 31A) FAK expression (p<0.001 for both) and phosphorylated p70s6k (p<0.001; Fig. 31D). There was a nonsignificant but large mean decrease in phosphorylated csrc (p=0.009; Fig. 31B). There were no statistical differences between groups for total csrc (p=0.579; Fig. 30C), total (Fig. 30D) and phosphorylated (Fig. 31C) ERK1/2 (p=0.605 and p=0.865, respectively) and total p70s6k (p=0.086; Fig. 30E). 99

101 Fig. 29 Non-significant increases in the 120 kda α7 integrin were seen in the rat gastrocnemius 56 d post-sci. There was no detection of the 70 kda α7 integrin. Representative immunoblots are shown in reference to the 43 kda Ponceau stain. Data are presented ± SD. Fig. 30 SCI can affect expression of proteins of the focal adhesion in the rat gastrocnemius. A) No significant differences were seen for the β1 integrin subunit. B) Total FAK expression was decreased 56 d post-sci. There were no changes in the total expression of C) csrc, D) ERK1/2 or E) p70s6k. F) Representative Western immunoblots shows large differences in FAK expression. All proteins are shown in reference to a 43 kda Ponceau stain to demonstrate uniform protein loading. ( ) denotes elevated expression (p<0.005) compared to SCI. Data are presented ± SD. 100

102 Fig. 31 Protein phosphorylation of signaling proteins of the focal adhesion were affected following SCI. Expression of A) phosphorylated FAK was decreased following SCI. B) phosphorylated csrc had a large, but non-significant, decrease in the SCI group compared to the SHAM group. C) There was no change in ERK1/2 phosphorylation. D) Drastic decreases in p70s6k activation following SCI. E) Representative images of Western blots showing decreased expression of proteins following SCI. ( ) denotes increased expression (p<0.005) compared to SCI. Data are presented ± SD. 101

103 Chapter 5: Discussion 102

104 The three projects completed were selected to investigate whether the focal adhesion has the potential to react to different cellular stresses. Certainly the loss of focal adhesion proteins in clinical cases such as SCI may have a role in injury-related muscle loss. Changes to the focal adhesion with aerobic exercise training, independently of or alongside APAP supplementation, could have a role in the beneficial response of skeletal muscle to loading and passive muscle manipulation using IASTM could target the mechanosensory properties of the focal adhesion complex. The results that were found, for the most part, fell in line with our hypotheses. The exception was the unexpected changes we saw with APAP supplementation. The role of IASTM and its capability of inducing integrin-dependent changes has not been investigated. Passive tension created by 10 min of manual massage has been shown to increase FAK and ERK1/2 phosphorylation in the vastus lateralis immediately after massage but with a return to baseline 2.5 h later (48). In accordance with this, our timepoints clearly demonstrate that there is no longterm response of the α7β1 integrin following roughly the same amount of muscle manipulation. Perhaps a longer duration of treatment (~30-60 min) may be enough passive stress to alter the integrin pathway. Another reason why there may not have been any difference is fiber type. The α7β1 integrin and focal adhesion are more heavily localized in type 1 muscle fiber (57; 70). Localized treatment of the soleus would need higher than clinical levels of pressure through the gastrocnemius. This would have been extremely uncomfortable for our subjects. Although we did not do a myosin heavy chain analysis on our sample, it is likely that the gastrocnemius was close enough for comparison to the vastus lateralis based upon activity levels of our subjects (151). Because of the lack of change anticipated for FAK, it is not 103

105 surprising that there were no differences in the downstream proteins. There is evidence that cycles of passive stretch in myotubes (82) and rats (20) can cause changes in ERK1/2 in a mechanotransduction-dependent manner, however the timepoints measured were all within 30 min of the end of stretch. Most studies that have noted changes in integrin or FAK expression have used stresses quite dissimilar to clinical massage pressures. Surgical ablation following unloading (70; 115), chronic overload (56), 30 min (19; 105; 181) and 90 min (Graham, in review) of downhill running, or basic aerobic or resistance training (164) most likely place substantially more stress on the passive capabilities of the muscle. It is unlikely that we created enough stress to cause muscle damage. However, this is the first study to our knowledge that has investigate csrc in human skeletal muscle. The changes in the soleus seen in the α7β1 integrin following 8 wk of APAP supplementation were quite surprising. The increase in the 70 kda α7 integrin and FAK in the APAP groups provide evidence that there could be changes in content or functionality of the ECM. In these same rats, there is an increase in collagen content but decreases in collagen cross-linking (Carroll, personal communication). The increase in collagen could be a driving signal for the increase in FAK phosphorylation. In mesangial cell culture, incubation of type I collagen, which is found in fibrillar form in the ECM of muscle, has been shown to increase FAK phosphorylation while collagen IV, which is present in the basement membrane, had no effect on FAK (2). Decreases in tensile strength of the collagen network of the ECM may induce changes with other structural ligands of the ECM, such as fibronectin or laminin. Laminin is a positive regulator of the α7 integrin as lack of laminin, either caused by genetic knockouts in mouse 104

106 models or genetic disruptions in humans (79), decreases α7 integrin expression. During muscle development the α7 integrin does not appear until after a laminin layer has began to assemble (64). To maintain sarcolemmal integrity, laminin may be upregulated, leading to an increase in α7 integrin and downstream proteins. The increase in expression of total and phosphorylated FAK could induce several changes to the cell, both of which we investigated. One potential scenario could be FAK-driven increases in hypertrophic signaling through the Akt pathway. Our increases in total p70s6k and our large, but not significant, increase in phosphorylated p70s6k could be an effect of FAK activation. However, the increase in FAK phosphorylation may direct increases in p70s6k activation independent of Akt through phosphorylation-induced inhibition of TSC2 (49). Inhibition of TSC2 would activate mtor and, in turn, p70s6k. Investigation of the role of Akt and TSC2 in response to APAP supplementation would help define which FAK pathway is being directed towards p70s6k. The FAK/cSrc complex and its role in skeletal muscle has been sparsely investigated. With no changes seen in ERK1/2, the increase in csrc activation may be signaling through the Ras/Raf or RhoA pathways, pathways that maintain the actin and tubulin cytoskeleton. Our lack of ERK1/2 change reinforces our opinion that FAK is acting either through the PI3K/Akt pathway or inhibiting TSC2. PGF2α has been shown to work through a PI3K/ERK1/2 pathway in skeletal muscle myotubes (110). Our evidence shows that in rats that is not the case as our ERK1/2 levels were unchanged. The atrophic signaling molecules IL-6 and Murf-1 have decreased mrna expression following chronic APAP supplementation compared to placebo, providing another mechanism for an increase in hypertrophic signaling (155). More evidence is needed as increases in mrna may not translate into increases in protein expression. 105

107 The results found for the gastrocnemius were interesting as well. We expected no change because the recruitment for this muscle during aerobic exercise, especially after 8 wk of training, would not be enough to create a large response. While we saw large non-significant increases in FAK and ERK1/2 phosphorylation in the exercise groups, while not expected, it is not necessarily surprising as there is evidence to changes in these proteins following aerobic exercise training (6; 164). More interestingly, there was a decrease in expression of phosphorylated p70s6k in the APAP groups. Why these differences in fiber type occur are unknown but the differences in prostaglandin receptor sensitivity or differing expression of the COX isoforms in the respective fiber types may be reasons for these differences.. The changes that were seen in SCI rat gastrocnemius were mainly in line with our expectations. Our decreases in FAK and p70s6k support the literature where there is evidence for decreases in FAK following unloading (50; 67; 70; 101) and overall decreases in protein synthesis that are targeted from unloading through the PI3K pathway (50; 67). The lack of change in the β1 integrin was not unexpected at our timepoints. Unloading seems to maintain total β1 integrin expression for 3 d in rats (115). In humans β1 expression increases 10 d after unloading then returns to baseline after 34 d (101). Our analysis only looked at total β1 integrin concentration. There may have been changes in the shift in expression from the mature β1d integrin to the undifferentiated β1a integrin, which has been shown to occur following injury (88). The increase, while not significant, in the α7 integrin is interesting. It is possible that there was an increase in satellite cell proliferation as a response to atrophying muscle. α7 integrin is highly upregulated following satellite cell activation (15). However, there is no evidence that has looked 106

108 at the role of satellite cells during the initial onset of SCI. In young males with an average of 9 y post-sci, satellite cell content was decreased in both fiber types (160). This is not surprising considering the amount of muscle turnover that would occur over 9 y. In our case, ERK1/2 doesn t seem to be involved in the atrophy signal for SCI although it is necessary for growth, maintenance and regulation of muscle myotubes (140) and can be directly related to sarcolemmal tension (112). The large decrease, while not statistically significant, in active csrc is a reflexion of activated FAK, although the stability of total csrc shows that there are perhaps elements outside of the focal adhesion that direct csrc expression. In conclusion, our evidence provides a model in which SCI and APAP administration can affect the focal adhesion pathway (Fig. 5.1). There needs to be more research in the underlying causes of APAP-induced changes to skeletal muscle such as with the basement membrane and ECM. The timecourse for integrin-mediated changes following SCI need to be further researched to gain an understanding of the differential regulation of atrophy. IASTM may create changes in the focal adhesion signaling complex, but more research into the amount of pressure, duration of treatment and fiber type response are necessary. 107

109 Figure 5.1. Summary of the main affectors of the integrin pathway in skeletal muscle. Activities that create an increase in integrin signaling are APAP supplementation in type 1 muscle fibers or activities that create stress along the sarcolemma. Unloading or injury, APAP supplementation in type 2 muscle fibers and passive muscle manipulation via IASTM have no effect or decrease integrin signaling. 108