TRANSLATION INITIATION SIGNALING COMPONENTS ALTERED BY MECHANICAL LOAD DICTATE SKELETAL MUSCLE HYPERTROPHY DAVID L. MAYHEW

Transcription

1 TRANSLATION INITIATION SIGNALING COMPONENTS ALTERED BY MECHANICAL LOAD DICTATE SKELETAL MUSCLE HYPERTROPHY by DAVID L. MAYHEW MARCAS BAMMAN, MENTOR J. EDWIN BLALOCK STUART FRANK W. TIMOTHY GARVEY TIMOTHY NAGY PAUL GOEPFERT, ad hoc A DISSERTATION Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy BIRMINGHAM, ALABAMA 2010

2 Copyright by David L. Mayhew 2010

3 TRANSLATION INITIATION SIGNALING COMPONENTS ALTERED BY MECHANICAL LOAD DICTATE SKELETAL MUSCLE HYPERTROPHY DAVID L. MAYHEW PHYSIOLOGY AND BIOPHYSICS ABSTRACT The regulation of protein synthesis (i.e., mrna translation) is an energetically costly and extensively regulated process, which is primarily regulated at the initiation step. Mechanical load is a potent stimulus of translation in skeletal muscle, and thus this tissue provides an excellent model system to study the plasticity of the translational apparatus. We investigated the effects of alterations in translation initiation cell signaling pathways in skeletal muscle using a variety of in vitro and in vivo mouse and human model systems. Collectively, our results suggest that, although many proteins in translational signaling pathways are responsive to mechanical load, the response of most were congruent with neither direct measures of muscle protein synthesis rates nor the degree of mechanical load-induced myofiber hypertrophy in humans. Of 11 translational signaling components examined, only p70s6k (T421/S424) phosphorylation and eif2bε abundance after acute mechanical loading were associated with eventual myofiber hypertrophy. Subsequent studies therefore focused on these two proteins. We found that overexpression of eif2bε was sufficient to increase cap-dependent protein synthesis rates in myogenic cells in vitro. Expression of eif2bε in mouse skeletal muscle in vivo was sufficient to increase myofiber size after only seven days. Although there was an in vivo association between p70s6k signaling and eif2bε abundance, genetic alteration of p70s6k signaling was not sufficient to increase eif2bε abundance in multiple cell lines in vitro or in mouse skeletal muscle in vivo. Overall, this series of experiments iii

4 demonstrates, for the first time, that eif2bε is an important determinant of myofiber size, and that augmentation of eif2bε abundance may largely dictate the degree of loadinduced myofiber hypertrophy. These studies provide mechanistic insight into eif2bεdependent regulation of myogenic cell size, which likely has important implications for cell size determination in all mammalian cells. iv

5 ACKNOWLEDGMENTS I would like to sincerely thank J. Edwin Blalock, PhD, Stuart Frank, MD, W. Timothy Garvey, MD, Timothy Nagy, PhD, and Paul Goepfert, MD for committing time from their busy schedules to further my education as members of my dissertation committee. I would like to extend my appreciation to both the students and administration of the Medical Scientist Training Program (MSTP) at UAB for financial, educational, and moral support. I would additionally like to thank my mentor Marcas Bamman, PhD for the opportunity to develop as a scientist while a member of his laboratory. His openness and willingness to explore new ideas created an intellectually stimulating environment that lead to this dissertation. Finally, I would like to thank my family for providing a supportive home environment, demanding the best from my efforts, instilling intellectual curiosity, and developing in me the discipline to investigate that curiosity. I would have neither begun nor completed this dissertation without their guidance and support. v

6 TABLE OF CONTENTS Page ABSTRACT... iii ACKNOWLEDGMENTS...v LIST OF TABLES... viii LIST OF FIGURES... ix CHAPTER 1 INTRODUCTION...1 Clinical significance of protein synthesis regulation...1 Diseases associated with dysregulated protein synthesis...1 Cellular hypertrophy...5 Two basic mechanisms of translation initiation...8 General mechanisms of cap-dependent translation initiation...11 Cell signaling in hypertrophy...14 Aims of dissertation TRANSLATIONAL SIGNALING RESPONSES PRECEDING RESISTANCE TRAINING-MEDIATED MYOFIBER HYPERTROPHY IN YOUNG AND OLD HUMANS...18 Abstract...19 Introduction...20 Methods...23 Results...29 Discussion...36 Grants and Acknowledgments...43 References eif2bε INDUCES CAP-DEPENDENT TRANSLATION AND SKELETAL MUSCLE HYPERTROPHY...49 Abstract...50 Introduction...51 Methods...55 Results...62 Discussion...75 Grants and Acknowledgments...80 References...81 vi

7 4 SUMMARY AND CONCLUSION...87 Signaling of most translation initiation components does not drive protein synthesis and hypertrophy...87 Potential mechanisms of p70s6k-induced myofiber hypertrophy...88 Potential mechanisms of eif2bε-induced myofiber hypertrophy...93 Overall model of mechanical load-induced myofiber hypertrophy...98 Concluding remarks and future directions LIST OF REFERENCES APPENDIX: IRB APPROVAL FORM vii

8 LIST OF TABLES Table Page TRANSLATIONAL SIGNALING RESPONSES PRECEDING RESISTANCE TRAINING-MEDIATED MYOFIBER HYPERTROPHY IN YOUNG AND OLD HUMANS 1 Baseline Descriptive Characteristics and Effects of Resistance Training on Lean Mass and Strength in Young and Old Subjects...35 viii

9 LIST OF FIGURES Figure Page INTRODUCTION 1 Regulation of eif4f Formation on Capped mrna Regulation of Translation Initiation by eif TRANSLATIONAL SIGNALING RESPONSES PRECEDING RESISTANCE TRAINING-MEDIATED MYOFIBER HYPERTROPHY IN YOUNG AND OLD HUMANS 1 Effects of an Unaccustomed Resistance Exercise Bout on Phosphorylation of Akt Effects of an Unaccustomed Resistance Exercise Bout on Phosphorylation of Eukaryotic Initiation Factor 4E (eif4e) Binding Protein (4EBP1) Effects of an Unaccustomed Resistance Exercise Bout on the Fractional Synthesis Rate (FSR) of Mixed Muscle Protein 24 h After Exercise in Young and Old Subjects Effects of a 16-wk Resistance Training Program on Type II Myofiber Cross-Sectional Area (CSA)...34 eif2bε INDUCES CAP-DEPENDENT TRANSLATION AND SKELETAL MUSCLE HYPERTROPHY 1 Resistance Exercise-Induced Changes in Translational Signaling Components not Altered Differentially by Hypertrophy response cluster Resistance Exercise-Induced Changes Translational Signaling Components that were Altered Differentially by Hypertrophy Response Cluster eif2bε Activity is Sufficient to Increase Cap-Dependent Translation in vitro Overexpression of eif2bε or eif2bε-s535a Induce Skeletal Muscle Fiber Hypertrophy p70s6k Signaling does not Alter Total eif2bε Cellular Abundance in vitro...71 ix

10 6 p70s6k Signaling does not Alter Total eif2bε Cellular Abundance in vivo...73 Figure Page SUMMARY AND CONCLUSIONS 1 Proposed Model for eif2b-dependent Myofiber Hypertrophy Proposed Model for Myofiber Hypertrophy Variability Seen Among Humans x

11 INTRODUCTION Clinical significance of protein synthesis regulation. The control of protein synthesis (i.e., mrna translation) rates is an important physiologic adaptation in many cell types in response to growth factor, nutrient, and mechanical stimulation. It is equally critical to the genesis and progression of diverse pathologic processes such as cardiovascular disease, cancer, polycystic kidney disease, leukoencephalopathies, and skeletal muscle atrophy, each of which are outlined in more detail below. Moreover, skeletal muscle atrophy adversely affects those suffering from a variety of medical conditions, including burns, cancer, diabetes, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), human acquired immunodeficiency virus (HIV), acquired immunodeficiency syndrome (AIDS), renal failure, and sepsis, and is exacerbated by normal aging and conditions leading to mechanical unloading or disuse (i.e. bed rest, cast immobilization, mechanical ventilation) (12, 63, 91, 92, 135). While significant strides have been made in the primary treatment of many of these diseases, treatment of the associated muscle loss has been largely ignored. This is especially true for age-related loss of muscle mass, termed sarcopenia. Since the ability to maintain muscle mass significantly contributes to favorable clinical outcome (63, 69), it is incumbent upon clinicians to be mindful of the degree of muscle mass loss secondary to the primary pathology/condition, and take steps to ensure minimal muscle atrophy. Diseases associated with dysregulated protein synthesis. Cardiovascular disease. Cardiovascular disease is the leading cause of death in the United States (62). Dysregulated hemodynamic patterns induce alterations in the protein synthesis (i.e., 1

12 translational) apparatus of the heart, which over time result in dilated cardiomyopathy and death. Pharmacological inhibition of this dysregulated translational activity in the pathologically remodeling heart resulted in improved cardiac structure and function (78, 137, 138). This suggests that translational dysregulation may at least partially underlie the pathologic myocardial hypertrophy that leads to dilated cardiomyopathy and associated morbidity and mortality. Moreover, inflammation is recognized as a hallmark of vascular dysfunction that results in atherosclerosis (95). This aberrant inflammation results in hyperplasia of vascular neointimal smooth muscle, causing coronary artery stent closure after angioplasty (24). Drugs that reduce protein synthesis rates, such as rapamycin analogs are now being used in drug eluting stents to delay stent closure and improve clinical outcome (49, 55). Cancer. Neoplastic transformation is the second leading cause of death in the United States (62). Dysregulated mrna translation was found to be associated with the transformed phenotype (11, 48, 119, 141). This may prove to be more critical during the progression from locally invasive disease to metastasis, as approximately 2-fold more transcripts were altered at the translational vs. transcriptional level in metastatic colorectal cancer (120). Dysregulated translation as a theme in cancer progression extends to treatment modalities as well, where ~10-fold more transcripts were altered on the translational level than on the transcriptional level in response to ionizing radiation (96). Although protein synthesis is increased in cancerous cells themselves, it is often concomitantly suppressed in the skeletal muscle of cancer patients, while muscle protein breakdown is increased, resulting in cancer-induced muscle atrophy (i.e., cachexia) that further exacerbates progression toward morbidity and mortality (145, 150). While much 2

13 effort has focused on anti-cancer drugs targeting the cell cycle, an increasing appreciation of the contribution of mrna translation to the neoplastic phenotype has resulted in the development of several chemotherapeutic agents that specifically target the translational apparatus (6, 14, 25, 34). Polycistic kidney disease (PKD). Autosomal dominant PKD (ADPKD) is a disease characterized by bilateral fluid filled cysts that generally develop in adulthood and is the most common genetic cause of kidney failure (47). This condition usually results from the mutation of either PKD1 or PKD2, which code for the polycistin-1 and - 2 proteins, respectively. These proteins normally provide a constitutively negative input to the mammalian target of rapamycin (mtor) pathway (31), a nexus of hypertrophic signaling (see below). Polycistin-1 and/or -2 signaling ablation results in constitutively high levels of mtor activity and protein synthesis, which is a prerequisite for excessive cellular proliferation. The unrestrained cell proliferation of renal tubular cells results in the fluid filled cysts characteristic of the disease. As with cardiovascular disease and cancer, promising treatment potential has arisen from the use of the protein synthesis inhibitor rapamycin (164), a macrolide antibiotic that inhibits mtor signaling (129). Leukoencephalopathy with vanishing while matter (VWM). Although the abovementioned diseases are associated with abnormally heightened protein synthesis, diseases also exist that suffer from the opposite phenomenon. One such disease is VWM. Symptoms of VWM include ataxia, mental decline, and spasticity, which occur due to the loss of white matter in the central nervous system (153). The severity and age of onset varies significantly, from severe childhood to mild adult manifestations. Moreover, onset or exacerbation of symptoms often occurs secondary to febrile illness or head trauma. At 3

14 least a subset of VWM cases are caused by mutations in any of the 5 subunits of the guanine nucleotide exchange factor (GEF) known as the eukaryotic initiation factor (eif) 2B (the role of eif2b in translation discussed below), with the majority of mutations occurring in the catalytic ε subunit (154), which reduces eif2b GEF activity (94). Since eif2b activity is required for protein synthesis initiating at an AUG codon, these mutations render cells susceptible to conditions that further reduce eif2b activity. In one study (74), although basal eif2b activity was not altered in primary or transformed fibroblasts from VWM patients, they exhibited a heightened stress response, consistent with the onset of symptoms in this patient population. There currently exists no treatment for this disease. Sarcopenia. Sarcopenia is a slow, degenerative disease of multifactorial yet poorly understood etiology characterized by marked declines in skeletal muscle mass and function with age (67, 105). Age-related muscle loss begins to occur in the fourth decade and gradually progresses throughout life, with losses of 1-2% per year beyond the fifth decade (71). Type II myofiber atrophy and losses of both type I and type II motor units, along with functional declines characteristic of sarcopenia, are associated with decreased mobility, increased likelihood of falls, decreased quality of life, increased rates of depression, and increased mortality from all causes (33, 70, 163). In the year 2000 the estimated direct health care costs due to sarcopenia totaled $18.5 billion in the U.S. (71), which will likely increase to a significant degree in the near future given the increasing average age of the population. Sarcopenia primarily arises from a reduction in skeletal muscle protein synthesis rates as opposed to an increase in protein degradation (79, 126, 161). Pharmacological measures to combat sarcopenia have been largely unsuccessful, 4

15 and resistance exercise continues to be the best known countermeasure to combat this condition (17, 41, 161). Clearly protein synthesis regulation has substantial implications in clinical medicine. While only cardiovascular disease, cancer, PKD, VWM, and sarcopenia have been briefly outlined above, many more exist. The purpose of this brief introduction to various translationally driven diseases was to provide an appropriate scope of the impact of dysregulated mrna translation on the patient population. This dissertation focuses more specifically on the regulation of muscle mass. Given that the decreased muscle mass accompanying many diseases and conditions often contributes to associated morbidity, mortality, and decreased quality of life (65, 163), it would appear crucial for health care professionals to address this phenomenon in conjunction with the primary pathologies. Characterizing the basic mechanisms of skeletal muscle cell size regulation will identify specific targets that can be addressed therapeutically. These treatments, when used in conjunction with current disease-specific treatments, will likely increase quality of life as well as decrease the deleterious consequences of numerous diseases and conditions that induce marked muscle atrophy, some of which are listed above. Cellular hypertrophy. Mechanical load is a potent hypertrophic stimulus to skeletal muscle. Although dramatic phenotypic changes occur in response to mechanical load, it alters the abundance of only a small subset of mrnas (26). While mechanical load-induced transcriptional alterations can and should not be overlooked, much work has recently focused on translational control in skeletal muscle hypertrophy (106). Translational control in muscle is typically compartmentalized into two temporally and 5

16 functionally discrete entities: translational efficiency and capacity (i.e., ribosomal biogenesis). Translational efficiency. Skeletal muscle hypertrophy is thought to occur primarily by an increase in protein synthesis (as opposed to decreased degradation), which is regulated at the level of translation initiation (81). This conclusion has come about from the observation that the amount of mrna engaged by multiple ribosomes increases in many acute hypertrophic model systems, while total mrna remains relatively constant (89). Additionally, pactamycin, a drug that prevents translation initiation but allows translation elongation on mrnas on which ribosomes have already engaged, also prevents increased protein synthesis in response to insulin (102) or mechanical load (7). Furthermore, ribosomal transit times, used as a surrogate for translation elongation, are equal between control and hypertrophied muscle (7, 102). Polysomal profiling has shown polysomal aggregation of mrna in response to resistance exercise in rat skeletal muscle, indicative of multiple ribosomes engaged on, and actively translating, mrna (89). Together these data indicate that the increased protein synthesis that results in myofiber hypertrophy is regulated at the level of translation initiation. Overall, cap-dependent translation initiation is regulated by two parallel processes: one involving the eukaryotic initiation factor (eif) 4F and the other involving eif2b (discussed in detail below). Translational capacity. In contrast to the acute regulation afforded by increased translational efficiency discussed above, a long-term increase in protein synthesis is driven by ribosomal biogenesis (100, 107). To accomplish this, cells increase the translation of a subset of mrnas that encodes ribosomal proteins, translation initiation 6

17 and elongation factors, and other growth promoting proteins, thereby increasing total translational capacity. Therefore, while ribosomal biogenesis is often thought of as distinct from translation initiation, it does require, and in fact is primarily regulated by, translation initiation itself (101). Many of these mrnas contain a common motif in their 5 -untranslated region (5 UTR) known as a 5 terminal oligopyrimidine tract (5 TOP) (160). 5 TOPs generally contain a C nucleotide followed by a stretch of 4-15 pyrimidines that are thought to confer translational repression upon their cis-regulated mrnas under non-growth conditions (8). It is through a variety of growth-promoting signals that these mrnas are de-repressed, and thus actively transcribed into proteins (23). Although the total number of 5 TOP-regulated mrnas was thought to be approximately 86, Yamashita et al. (160) recently used genome-wide database searches of their previously obtained sequence data to identify 1645 candidate TOP genes, many of which were further validated, thereby expanding the known proteins regulated by growth promoting signals at the translational level. Interestingly, they found that virtually all ribosomal proteins and elongation factors contain a 5 TOP, but they also discovered that other genes previously unidentified as 5 TOPs contain them as well, which include translation initiation factors and lysosome- and metabolism-related proteins. Given the identity of the newly identified 5 TOP genes, they proposed that proteins encoded by 5 TOP mrnas are likely to play key roles in directly or indirectly regulating a majority of cellular mrnas. Therefore, translational regulation of 5 TOP mrnas may represent a targeted mechanism by which the cell can coordinate ribosomal biogenesis and cellular hypertrophy. Again, the translation of 5 TOP-regulated mrnas occurs due to alterations in translational efficiency. Since translational efficiency is central to the hypertrophic 7

18 phenotype, this dissertation focuses on the impact of mechanical load on cell signaling pathways that affect translational efficiency. Two basic mechanisms of translation initiation. A substantial percentage of total cellular energy is devoted to maintaining protein synthesis. Such an energetically costly process demands extensive regulation so as to specifically tailor the response to cellular needs. Transcriptional activity of specific mrnas certainly contributes to the ultimate content of its cognate protein, however the final gatekeeper for protein synthesis is at the level of translation. Indeed, mrna content is a poor surrogate for protein synthesis/abundance since extensive regulation occurs at the level of translation (104, 148). While mrna translation is regulated at the initiation, elongation, and termination steps, the vast majority of the physiologically relevant regulation occurs prior to and during initiation (140). Two basic mechanisms of translation initiation exist: capdependent and internal ribosome entry site (IRES)-dependent. Cap-dependent vs. IRES translation. Both mechanisms of translation initiation utilize RNA binding proteins that coordinate the process of mrna recruitment to the 40S ribosomal subunit, the activies of which are regulated (positively or negatively) by a host of hormones, growth factors, cytokines, and environmental stresses. Cap-dependent translation represents the majority of cellular mrna translation (90-95%), and is therefore the focus of this dissertation. A more detailed description of cap-dependent translation and the signaling pathways that regulate its activity is presented below. Briefly, however, cap-dependent translation occurs when the ribosome engages the mrna molecule at the 5 -methyl guanosine cap of the mrna, which is aided by no fewer than 29 eif mrna binding proteins. Together with the 40S ribosomal subunit, 8

19 these proteins constitute the 43S pre-initiation complex. Once engaged on the mrna, the 43S complex scans from the 5 cap in the 3 direction through the 5 UTR. After the ribosome recognizes a start codon that is imbedded in a favorable Kozak consensus sequence (87), guanosine triphosphate (GTP) hydrolysis occurs, which promotes the dissociation of numerous mrna binding proteins and the recruitment of the 60S ribosomal subunit. Together the 40S and 60S subunits constitute a functional 80S ribosome, which translates the coding region of the mrna into protein. Under a favorable pro-growth environment, cap-dependent mrnas compete well for a limiting amount of translationally competent ribosomes. In other words, mrna content is in most cases not the rate limiting factor in translation initiation. Conversely, IRES-dependent mrnas compete less favorably under growth conditions. Under stress conditions, which initiate signaling events that reduce the number of functionally competent ribosomes, cap-dependent translation is severely compromised while IRESs compete more favorably. This is most likely due to stress-regulated association of IRESs with protein factors that bind to and affect their translation, termed IRES trans-activating factors (ITAFs) (22). Therefore, paradoxically, those mrnas regulated by IRESs may experience an increase in translational efficiency under conditions of reduced capdependent translation. IRES-driven translation occurs by a mechanism distinct from cap-dependent translation, and thus is subject to independent regulation. Generally IRES-regulated mrnas contain long, highly structured 5 UTRs, which impede cap-dependent translation and allow IRES translation to predominate for a particular transcript (143). However, all mrnas that are translated by an IRES-dependent mechanism are also translated by a 9

20 cap-dependent mechanism, although the degree of cap-dependent translation varies depending on the length and complexity of the 5 UTR. Although consensus primary sequences that confer IRES activity have not been identified, it is well accepted that secondary structures within the 5 UTR provide binding sites for ITAFs, which, analogous to eifs, recruit the mrna to the ribosome (142). IRES translation occurs in only 5-10% of cellular mrnas and is usually much less efficient than cap-dependent translation (143). As such, the products of IRES translation are generally needed in very small quantities (e.g., transcription factors). As stated above, the translation of most IRESs is maintained or upregulated in response to cellular stress when cap-dependent translation is severely compromised. This includes proteins that aid in stress recovery, such as p53, VEGF, c-myc, Bcl-2, c-iap, XIAP, HSP70, HSP90, eif4g, Bip, etc. Conversely, should stress be sustained or of sufficient magnitude, pro-apoptotic proteins such as Apaf-1 and DAP5 are translated by IRESs to aid in coordinated cell death, ultimately maintaining viability of the organism by removing cells that poorly accommodate stress. Both cap-dependent and IRES-dependent translation serve the same ultimate purpose: to recruit a functionally competent ribosome to the start codon of mrna to begin translation. However, IRES translation allows the cell to tailor the synthesis of specific proteins under distinct environmental stresses. Although this response has significant implications for overall cell physiology, IRES driven translation comprises only a small degree of overall translation and therefore will not be discussed further here. General mechanisms of cap-dependent translation initiation. eif4f regulation. As mentioned above, translation is controlled primarily at the step of initiation (81). Translation initiation is a multi-step process that is highly regulated. 10

21 Nascent cytosolic mrnas that contain a 5 -methylguanosine cap are bound by the eif4e, 4G, and 4A (together termed eif4f) (Figure 1). The formation of eif4f is partially controlled by mtor-mediated phosphorylation of the eif4e binding proteins (4E-BPs), the best characterized of which is 4E-BP1. When hypophosphorylated, 4E-BP1 exists in an inhibitory complex with eif4e. When 4E-BP1 is phosphorylated it releases eif4e, which allows eif4e to complex with eif4g and eif4a (AAA) n eif4g eif4a eif4f 7 Me-GTP AUG thus creating the eif4f complex. eif4e mtor 4E-BP1 P 4E-BP1 7 Me-GTP AUG Ribosomal Binding (see Figure 2) (AAA Figure 1. Regulation of eif4f formation on capped mrna. Availability of the cap binding protein eif4e is primarily regulated by mtormediated phosphorylation of 4E-BP1. eif4f is required for cap-dependent translation initiation (50), which includes all mrnas without an IRES. The eif4f-bound mrna is recruited to the 43S pre-initiation complex (also discussed below), resulting in the 48S complex. The creation of the 48S complex permits the subsequent binding of the large (60S) ribosomal subunit, thus creating a translation-competent ribosome. General eif2 regulation. In a series of events that are parallel to, but equally important as, eif4f regulation, eif2 shuttles the initiator trna (Met-tRNA i ) to the initiator codon on a nascent mrna, which is generally imbedded within a Kozak consensus sequence (AccAUGG). In this process the binding of eif2 to Met-tRNA i is dependent on GTP binding to eif2 (Figure 2). After the formation of eif2 GTP MettRNA i, termed the ternary complex, binding to the small (40S) ribosomal subunit can 11

22 occur and, along with binding of other factors, results in the 43S pre-initiation complex. The eif4f-bound mrna (discussed above) is shuttled to the 43S pre-initiation complex, creating the 48S complex, where eif2 GTP Met-tRNA i scans the mrna for the initiator codon. Once Met-tRNA i binds to the initiator codon in the P site of the 40S ribosomal subunit, GTP is hydrolyzed to GDP by eif2 aided by the GTPase activating protein (GAP) eif5. This induces release of eif2 GDP and other factors with subsequent binding of the large (60S) ribosomal subunit to the 40S mrna complex, GTP 40S Cap-bound mrna (See Figure 1) GTP GTP 7 Me-GTP AUG (AAA) n while Met-tRNA i Ternary Complex 43S Complex 48S Complex Scanning remains bound in the P GTP 7 Me-GTP AUG GTP (AAA) n site. Since the affinity of eif2 for GDP is at least eif2 eif2b GDP + P i GDP 60S 400-fold higher than for Translation Elongation 7 Me-GTP AUG (AAA) n GTP (80, 112), and the dissociation constant for GDP is low (112, 157), 80S Complex Figure 2. Regulation of translation initiation by eif2. Note that eif2b serves to recycle eif2 by catalyzing the exchange of GDP for GTP, allowing subsequent rounds of translation initiation. Adapted from Kapp & Lorsch (76). the exchange of GDP for GTP on eif2 must be catalyzed. This function is performed by the eif2b holoenzyme, which is composed of α-ε subunits. Relevant to this dissertation, the ε subunit serves as the catalytic subunit of the complex and is regulated by a variety of hormonal (21, 158), nutritional (51, 82), and mechanical (52) stimuli. The recycling of GDP for GTP is required for subsequent rounds of translation initiation; inhibition of eif2b activity results in severely impaired global protein synthesis (132). Guanine 12

23 nucleotide exchange factor (GEF) activity of eif2b is a point of regulation in a variety of scenarios, including mechanical load (86) and amino acid stimulation (82). Therefore, the coordinated regulation of both eif4f and eif2 is required for efficient translation initiation. eif4f vs. eif2 in cell size regulation. Much work has focused on the regulation of the eif4f complex, including eif4e and eif4g, in binding capped mrna and promoting translation initiation. However, we hypothesized that eif2b exerts a greater influence over myofiber hypertrophy than other initiation factors in response to mechanical load. This hypothesis is supported by previous work, where mechanical load resulted in alterations in neither eif4e nor eif4g phosphorylation or association (43, 44) while eif2bε activity was increased, which paralleled protein synthesis rates (43). Additionally, somatostatin treatment of rats, which reduces plasma insulin, prevented the leucine-induced increase in skeletal muscle protein synthesis, but did not prevent the leucine-induced formation of the eif4f complex (5). A parallel phenomenon was reported in rat liver, where after a fast rapamycin (a potent mtor complex 1 (mtorc1) inhibitor) administration blunted both the feeding-induced increase in total protein content and 5 TOP translation, but did not alter eif4e eif4g association or overall capdependent translation (4). A similar dissociation of eif4f regulation and protein synthesis was observed in L6 myoblasts, where eif4e 4E-BP1 and eif4e eif4g associations in response to insulin treatment were unaffected by amino acid withdrawal, whereas protein synthesis rates and eif2b activity decreased sharply (82). Similarly, while the insulininduced increase in protein synthesis was blocked by inhibitors of mtor, PI3K, or MEK1/2, these inhibitors had no effect on eif4e eif4g association (83). Together, these 13

24 data suggest that eif4f complex formation is not rate limiting under a variety of scenarios and that eif2b activity correlates with protein synthesis rates and cell size in vitro and in vivo. The importance of eif2bε in translation and cell size regulation was perhaps best exemplified by large increases in cardiac myocyte protein synthesis and cell size (as measured by surface area and protein/dna content) induced by expression of a constitutively active (non-phosphorylatable) eif2bε, while a dominant negative eif2bε mutant prevented isoproterenol-induced protein synthesis and cell hypertrophy (59). Further, a dominant negative upstream regulator of eif2bε, GSK3β, resulted in striking atrophy of C2C12 myoblasts in vitro (127). Therefore, it seems that eif2b-mediated GTP recycling on eif2, but not eif4f-mediated 5 -capped mrna binding, may be more critical to the in vivo translational response to growth-promoting stimuli, and may implicate eif2bε as a rate limiting factor. Indeed, the relative expression of eif2bε compared to other initiation factors suggests that it may be rate limiting (82, 139). The data presented in this dissertation are supportive of this conclusion, and provide the first evidence to date that eif2bε regulates cell size in vivo. Cell signaling in hypertrophy. mtor Signaling. Since cellular hypertrophy is dependent on translation initiation (81), an overview of the mechanical load-induced pathways that control translation initiation is relevant. The mtor signaling pathway is thought to be a nexus of translational control in response to growth factors (127), nutrient availability (58), mechanical stimuli (66, 89), and cellular energy status (30). Thus the cell must reside in a nutrient-, energetic-, and growth factor-favorable environment in order to actively initiate translation of cellular proteins. mtor exists in two distinct 14

25 complexes, termed mtor complex 1 (mtorc1) and 2 (mtorc2). Both mtor complexes contain as their core proteins mtor and GβL. While mtorc1 contains a protein known as regulatory associated protein of TOR (raptor), mtorc2 contains the rapamycin insensitive companion of TOR (rictor), each of which provides downstream substrate specificity for each respective mtor complex. mtorc2 mediates actin cytoskeletal dynamics through poorly understood mechanisms (159) and will not be discussed here. mtorc1 controls translation initiation through its actions on at least two downstream targets, 4E-BP1 and ribosomal protein S6 kinase 1 (p70s6k) (45). mtorc1 binds to downstream substrates through a direct interaction of raptor with the TOR signaling (TOS) motif located in target proteins, which is required for rapamycinsensitive, mtor-dependent phosphorylation (108). Both p70s6k and 4E-BP1 have been shown to contain TOS motifs (130), and are phosphorylated by mtor in vitro (19, 131). p70s6k is the mtor effector of hypertrophy. Much effort has gone into evaluating mtor-dependent phosphorylation of 4E-BP1 and thus eif4e availability in an assortment of normal and diseased tissues, including muscle. Although some have observed eif4e (45) and 4E-BP1 (131) to affect cell size when expression was experimentally manipulated in various cell lines, others have found that mouse skeletal muscle mass correlated poorly with eif4e content as well as 4E-BP1 content and phosphorylation (111). Recently, the the binding of the 4E-BPs to eif4e was found to inhibit proliferation but not cell size (35). Conversely, it has been reported that p70s6k signaling exerts a substantial influence over cell size in skeletal muscle (9), and does so with greater proclivity than in other tissue types (111). p70s6k phosphorylation has been shown to correlate with myofiber hypertrophy in a fiber type (85), muscle type (9), and 15

26 contraction type (40) specific manner. Further, knockout (111, 117), knockdown (125), and overexpression (45) studies have linked p70s6k kinase activity to cell size control in a causative manner. Moreover, IGF-I treatment of C2C12 myotubes, which results in cellular hypertrophy, was dependent on p70s6k signaling but did not alter 4E-BP1 phosphorylation (114). Although an inactivating mutant of p70s6k (T389A) decreased overall cap-dependent translation, the binding of eif4g, eif4e, or 4E-BP1 to a 7-methyl- GTP cap analog complex was found to be unaltered (64), indicating that p70s6kdependent translational regulation of capped mrna occurs at a level other than eif4f mrna binding. However, p70s6k signaling stimulates the phosphorylation of eif4b, which promotes the helicase activity of eif4a to unwind complex mrna secondary structures (134). Therefore, although p70s6k does not seem to alter eif4f formation, it may promote helicase activity after mrna binding has occurred. Recently a newly identified p70s6k target known as S6K1 Aly/REF-like target (SKAR) was found to at least partially account for the effects of p70s6k signaling on cell size regulation (125), however the mechanism of SKAR-dependent cell size regulation is unknown. Therefore, the specific mechanism(s) of p70s6k-induced cellular hypertrophy are not fully realized. Together, these data indicate that while there is some degree of cell signaling component specificity in the tissue-specific regulation of cell size, p70s6k garners greater relative importance than eif4e or 4E-BP1 in skeletal muscle when compared to other tissue types. Therefore, of the two branches of the mtor pathway, p70s6k likely plays a greater role in the regulation of in vivo myofiber size than does 4E-BP1 and eif4e. Aims of dissertation. Skeletal muscle is arguably the most mechano-responsive tissue in the human body. It is exquisitely tuned to sense mechanical stimuli and to 16

27 respond by coordinating structural, functional, and metabolic responses to dampen the effects of future mechanical insults (i.e., overcompensation). The majority of the regulation of protein synthesis occurs at the level of mrna translation initiation (7, 102). Skeletal muscle is thus an excellent model system to study the plasticity of the translational apparatus. We thus chose to investigate the effects of mechanical load on the translational apparatus in skeletal muscle using in vitro and in vivo approaches. As discussed above, protein synthesis profoundly affects the phenotype of skeletal muscle. In humans this results in alterations in muscle mass and strength that ultimately affects mobility and overall quality of life, particularly in elderly (163). Thus, deciphering the mechanisms by which human skeletal muscle cell size regulation occurs will provide significant insight into potential therapeutic modalities that can result in increases in muscle mass and function. The effects of such interventions would ultimately be realized in reduced morbidity and mortality, increased quality of life, and reduced health care costs for a significant portion of the general public (33, 71, 163). This dissertation attempts to address the mechanisms by which skeletal muscle regulates mechanical load-induced increases in myofiber hypertrophy in humans. To do this we used a multilayered approach, with human skeletal muscle tissue as the primary, initial model system. We then chose to examine phenomena observed in human subjects in more detail by utilizing both in vitro and mouse in vivo systems in an attempt to gain mechanistic insight into the hypertrophic phenotype. Through these targeted approaches, we have identified and characterized eif2bε as an important determinant in regulating myofiber hypertrophy in vivo. 17

28 TRANSLATIONAL SIGNALING RESPONSES PRECEDING RESISTANCE TRAINING-MEDIATED MYOFIBER HYPERTROPHY IN YOUNG AND OLD HUMANS by DAVID L. MAYHEW, JEONG-SU KIM, JAMES M. CROSS, ARNY A. FERRANDO AND MARCAS M. BAMMAN Journal of Applied Physiology [ /japplphysiol ] Copyright 2009 by The American Physiological Society Used by permission Format adapted for dissertation

29 ABSTRACT While skeletal muscle protein accretion during resistance training (RT)-mediated myofiber hypertrophy is thought to result from upregulated translation initiation signaling, this concept is based on responses to a single bout of unaccustomed resistance exercise (RE) with no measure of hypertrophy across RT. Further, aging appears to affect acute responses to RE, but whether age differences in responsiveness persist during RT leading to impaired RT adaptation is unclear. We therefore tested whether muscle protein fractional synthesis rate (FSR) and Akt/mammalian target of rapamycin (mtor) signaling in response to unaccustomed RE differed in old vs. young adults, and whether age differences in acute responsiveness were associated with differences in muscle hypertrophy after 16 wk of RT. Fifteen old and 21 young adult subjects completed the 16- wk study. The phosphorylation states of Akt, S6K1, ribosomal protein S6 (RPS6), eukaryotic initiation factor 4E (eif4e) binding protein (4EBP1), eif4e, and eif4g were all elevated (23 199%) 24 h after a bout of unaccustomed RE. A concomitant 62% increase in FSR was found in a subset (6 old, 8 young). Age x time interaction was found only for RPS6 phosphorylation (+335% in old subjects only), while there was an interaction trend (p = 0.084) for FSR (+96% in young subjects only). After 16 wk of RT, gains in muscle mass, type II myofiber size, and voluntary strength were similar in young and old subjects. In conclusion, at the level of translational signaling, we found no evidence of impaired responsiveness among older adults, and for the first time, we show that changes in translational signaling after unaccustomed RE were associated with substantial muscle protein accretion (hypertrophy) during continued RT. 19

30 INTRODUCTION The long-term net balance of skeletal muscle protein synthesis and breakdown rates significantly influences whole body protein balance and determines the state of muscle mass. A net negative balance over an extended time period, whether due to suppressed synthesis or upregulated breakdown, leads to muscle atrophy, which plays an important role in health status for a variety of disease states, including HIV/AIDS, cancer, renal failure, sepsis, diabetes, aging, bed rest, and failure to wean from the ventilator (27, 28, 41). Unlike acute muscle wasting (e.g., sepsis, burns), which is measurable in days and is caused by hypercatabolism, the slow muscle atrophy of aging (i.e., sarcopenia) is only measurable across extended periods (e.g., several years) and is thought to result, at least in part, from impaired or slowed regenerative and growth processes consequent to anabolic stimuli. It is therefore of clinical importance to manipulate synthesis rates toward the preservation and/or accretion of muscle protein in older adults. Resistance exercise training (RT) is a well-established means of inducing muscle hypertrophy by coordinating a net gain in mixed muscle protein. This apparently results from acute upregulation of inward amino acid transport (5) leading to an elevated fractional synthetic rate (FSR) of muscle protein for as many as 48 h following each exercise bout (5, 8, 17, 48). Resistance exercise (RE) also acutely increases the fractional breakdown rate (FBR) (5, 36), but over time, net synthesis must occur for measurable hypertrophy, and the balance between FSR and FBR is dependent on feeding state (5, 34, 36) as well as training status (34, 36). RT induces hypertrophy of all myofibers and because the adaptation is preferential to type II myofibers (25), it is seemingly the ideal treatment to counteract the type II atrophy characteristic of aging sarcopenia. However, 20

31 the efficacy of RT-mediated hypertrophy is highly variable across human subjects (3) and tends to decline with advancing age, particularly in old vs. young men (25, 31). This warrants a better understanding of the mechanisms regulating RT-mediated increases in FSR and eventual hypertrophy and the influence of age on these processes. Some of the chief intracellular signaling pathways involved in skeletal muscle hypertrophy are beginning to be studied in detail. The regulation of translation initiation via the PI3K/Akt/mammalian target of rapamycin (mtor) pathway is recognized as a significant regulator of muscle mass, and excellent reviews have been provided elsewhere (30, 39). This pathway has been shown to be upregulated in multiple hypertrophic model systems both in vitro (40) and in vivo (6). Key downstream targets of the kinase mtor include 1) the eukaryotic initiation factor 4E (eif4e) binding protein (4EBP1), which on phosphorylation releases its inhibition over eif4e to promote 5'-methylguanosine capdependent translation initiation; and 2) p70 S6 kinase (S6K1), which is a known modulator of cell size. Characterizing alterations of these signaling processes and how they may influence muscle protein synthesis rates in response to mechanical load is important for devising effective therapeutic interventions to promote muscle hypertrophy or muscle regrowth following atrophy (e.g., aging sarcopenia). However, to date no studies have examined the influence of aging on content/activation of proteins in this signal transduction pathway in conjunction with in vivo protein metabolism in response to acute RE, nor during long-term RT. The aims of this research were therefore to assess, in young vs. older adults, human skeletal muscle protein metabolism and translation initiation signaling in response to a single bout of 21

32 unaccustomed RE and subsequent myofiber hypertrophy after a long-term RT program. We tested the following hypotheses: 1) young muscle is more responsive to unaccustomed RE than old based on a more robust increase in muscle protein FSR and upregulation of translation initiation signaling; and 2) age differences in translational signaling responsiveness to RE will lead to age differences in hypertrophy after repeated exposures during RT (16 wk x 3 days/wk). Thirty-six subjects (21 young, 15 old) were tested for responsiveness to unaccustomed RE, and all 36 then completed a 16-wk resistance training program. 22

33 METHODS Subjects. Thirty-six untrained but otherwise healthy adults (21 young; 15 old) were recruited from the Birmingham, Alabama, metropolitan area for participation in this research as part of a larger 16-wk exercise training clinical trial. Because a major aim of this work focused on translational signaling, we determined the young vs. old sample size based on effect sizes in prior work in which we found a significant age x time interaction in MAP kinase signaling with 17 young and 13 old adults (24). All subjects completed health history and physical activity questionnaires. Older adults passed a comprehensive physical exam conducted by a geriatrician and a diagnostic, graded exercise stress test with 12-lead ECG reviewed by a cardiologist. Subjects were free of any musculoskeletal or other disorders that might have affected their ability to complete resistance training and testing for the study. Subjects were not obese (body mass index < 30), and none had undergone knee extensor resistance training within the past 5 years. None of the subjects were being treated with exogenous testosterone or other pharmacological interventions known to influence muscle mass. The study was approved by the Institutional Review Boards of both the University of Alabama at Birmingham (UAB) and the Birmingham Veterans Affairs Medical Center. Written informed consent was obtained before participation in the research. Unaccustomed resistance exercise bout and progressive resistance training. Following familiarization, baseline one-repetition maximum (1RM) strength was evaluated on squat, leg press, and knee extension exercises as described (4, 33). Participants then completed 2 days of training familiarization followed by the first full RE 23

34 bout, consisting of three sets at 8 12 RM with 90-s recovery between sets on each of the three exercises. This first bout represented the first session of a 3 days/wk, 16-wk progressive RT program. Resistance loads were increased when a subject could perform 12 repetitions in two of the three sets for a given exercise. 1RM strength was reevaluated after 8 and 16 wk. All training sessions were supervised by a Certified Strength and Conditioning Specialist (National Strength and Conditioning Association) and/or Certified Health Fitness Instructor (American College of Sports Medicine). Lean mass. Thigh lean mass and total body lean mass were determined by dualenergy X-ray absorptiometry (DEXA) using a Lunar Prodigy (model no. 8743, GE Lunar, Madison, WI) and encore 2002 software (version ) according to manufacturer's instructions. Analyses at baseline and after 16 wk of RT were performed by the same technician blinded to intervention time point. Muscle tissue collection. Muscle tissue was removed under local anesthetic (2% lidocaine) from vastus lateralis by percutaneous needle biopsy using a 5-mm Bergstrom biopsy needle under suction as previously described (13), with any visible fat or connective tissues dissected at the bedside. Muscle tissue samples were collected at baseline, 24 h after the first full exercise bout (acute response), and 24 h after the last training bout at week 16. At baseline and week 16, 70 mg were mounted crosssectionally in liquid nitrogen-cooled isopentane for subsequent histological analysis, and the remainder was weighed, divided, and snap-frozen (25 35 mg/tube) in liquid nitrogen. All tissue samples collected at the acute response time point were snap-frozen. Samples (25 mg) used for muscle protein synthesis measurements (see Muscle protein synthesis) 24

35 were briefly rinsed with ice-cold saline to remove residual blood and fluid before being snap-frozen. Myofiber size and distribution. Using immunofluorescence microscopy techniques described previously (21, 25), myofibers positive for myosin heavy chain (MHC) type I (MHCI) and negative for MHCIIa were classified as type I, fibers positive for MHCIIa and negative for MHCI were classified as type IIa, and fibers negative for both MHCI and MHCIIa were classified as type IIx. Hybrid myofibers (e.g., coexpression of I/IIa or IIa/IIx) that were revealed by both color and intensity using this technique were excluded from analyses. Myofiber type distribution and size were determined in blinded fashion by a single analyst as described (21, 25). For cross-sectional area (CSA) measurements, each myofiber was manually traced along its laminin-stained border. Myofiber size was measured for a minimum of 50 randomly selected myofibers per type, and type distribution was assessed on an average of 1,067 and 888 myofibers at baseline and week 16, respectively. One older subject was prescribed an anticoagulant during the final few weeks of resistance training; thus a 16-wk biopsy was contraindicated. For one young subject, the histological specimen at week 16 did not satisfy our analysis criteria. Consequently, pre- and posttraining histological specimens for 34 of 36 subjects were analyzed, yielding histological data on 20 young and 14 older subjects. Translation initiation signaling. The content and phosphorylation of putative proteins involved in translational signaling were assessed in 35 subjects (20 young, 15 old) at baseline and 24 h after the first bout of unaccustomed resistance exercise to determine whether aging influenced the single-bout response. Standard immunoblotting 25

36 was performed using established methods in our laboratory (4, 24). Muscle protein lysate was extracted from frozen muscle samples (average mg) as detailed previously (4, 24, 25). Protein concentrations were determined using the bicinchoninic acid (BCA) technique with BSA as a standard. Samples were run on 4 12% Bis-Tris (Invitrogen) SDS-PAGE gel matrixes with 35 µg total protein loaded into each well, which was determined as ideal by preliminary experiments. Samples within subjects across time were loaded in adjacent lanes. Proteins were transferred to PVDF membranes at 100 ma for 12 h. Primary antibodies against phospho(s473)- and total Akt; phospho(s2448)- and total mtor; phospho(t389)-, phospho(t421/s424)-, and total S6K1; phospho(s240/244)- and total ribosomal protein S6 (RPS6); phospho(t37/46)- and total 4E-BP1; phospho(s209)- and total eif4e; and phospho(s1108)- and total eif4g were purchased from Cell Signaling Technologies (Danvers, MA). Ideal primary antibody dilutions were determined by preliminary experiments and were 1:250 (vol/vol) for S1108-eIF4G, and 1:1,000 for all other antibodies. Horseradish peroxidase (HRP)- conjugated secondary antibody was used at 1:50,000 (wt/vol) followed by chemiluminescent detection in a Bio-Rad ChemiDoc imaging system with band densitometry performed using Bio-Rad Quantity One software (version 4.5.1). Parameters for image development in the ChemiDoc were consistent across all membranes using predefined saturation criteria for the CCD camera. Equal protein loading was verified by Ponceau S staining in most cases (not shown). Muscle protein synthesis. Stable isotope tracer infusion procedures were employed to assess the fractional synthetic rate (FSR) of vastus lateralis mixed muscle protein. These were performed as inpatient procedures on the UAB Pittman General 26

37 Clinical Research Center (GCRC). Subjects were admitted to the GCRC the evening prior and were provided a standardized meal. The infusion protocol began at 0600 after 8 10 h of fasting. Subjects received a 5-h primed (2 µmol/kg), continuous (0.05 µmol kg 1 min 1 ) infusion of L-[ring- 2 H 5 ]phenylalanine (d 5 -PHE; Cambridge Isotope Labs) via an antecubital venous catheter for determination of FSR. Isotopic steady state in the free amino acid pools in blood and muscle, which is accomplished in 2 h, was required to calculate protein kinetics via the precursor-product method (35). d 5 -PHE infusion began at 0 h and continued for 5 h. At 2 h, the first biopsy was taken under local anesthetic to measure the isotopic enrichment of d 5 -PHE in the intracellular free and incorporated (into full-length proteins) pools. The muscle incorporated protein enrichment of d 5 -PHE at 2 h served as the first time point for FSR measurement, while the enrichment at 5 h served as the second time point. FSR was therefore determined as the rate of tracer incorporation from the intracellular pool into the incorporated muscle protein fraction. Free amino acids were isolated from serum, intracellular, and protein-incorporated fractions using filtered prep columns with AG 50W-X8 resin and established laboratory techniques. Measurement of d 5 -PHE tracer enrichment of each fraction was accomplished by gas chromatography mass spectrometry (GCMS). FSR was calculated as described previously (35) and expressed as percent per hour. FSR was assessed at baseline and 24 h after the unaccustomed resistance exercise bout in 15 (8 young, 7 old) of the 36 subjects. Postexercise FSR was not valid for one of the older subjects; thus n = 8 young and 6 old subjects. Statistics. Age x time repeated-measures ANOVA was used to test main effects of time and age, and age x time interactions. For acute responses to a single bout of 27

38 resistance exercise, data were tested using two (age group) x two (time) repeatedmeasures ANOVA with baseline and acute response time points. Across 16 wk of training, data were tested via ANOVA with repeated measures as follows: 1RM strength, two (age group) x three time points (baseline, week 8, week 16); myofiber size and type distribution, two (age group) x two time points (baseline, wk 16). Tukey HSD tests were performed post hoc. Data are presented as means ± SE. Significance was accepted at p < 0.05 for all tests. 28

39 RESULTS Acute responses to unaccustomed resistance exercise. Results for the translation initiation signaling targets assessed are shown in Figs. 1 and 2. A noteworthy overall summary of the acute response translational signaling results is that only one significant age x time interaction was found (phosphorylation of RPS6). On the other hand, significant main time effects were noted for nearly all targets assessed throughout the signaling cascade, indicating that translational signaling was upregulated irrespective of age at the 24-h postexercise time point. Akt phosphorylation was increased 51% (p < 0.001, Figure 1A) while total Akt protein content was elevated 20% (p < 0.005). Within age groups, phosphorylated Akt was increased 60% among young and 39% among old subjects with no age x time interaction (p = 0.37). No changes in total or phosphorylated mtor were found. Downstream of mtor, we were unable to detect measurable phosphorylation of S6K1 at T389 (data not shown), which is thought to be largely an mtor-specific site (19), most likely due to the fasted state of the subjects. However, phosphorylation of the S6K1 autoinhibitory domain (T421/S424), which is driven primarily by ERK1/2 and p38 signaling (20, 45), was enhanced 61% overall (p < 0.05) (Figure 1B) while total S6K1 did not change. A concomitant and robust 199% increase (p < 0.001) was found for RPS6 phosphorylation (S240/S244) overall (Figure 1C), while total RPS6 was unchanged. An age x time interaction for RPS6 phosphorylation (p < 0.05) was driven by a significant 335% increase among old. Despite the lack of T389 phosphorylation of S6K1, increased phosphorylation was noted for another mtor downstream target, 4EBP1. The phosphorylation state of 4EBP1 was elevated 23% (p < 0.05), an effect primarily driven by young subjects (Figure 2A). At the level of the 29

40 Figure 1. Effects of an unaccustomed resistance exercise bout on phosphorylation of Akt (A), S6K1 auto-inhibitory domain (T421/S424) (B), and ribosomal protein S6 (RPS6) (C) 24 h after exercise (Post) in young and old subjects. D: representative immunoblots. Bars are means ± SE. Significant main effects and interaction (Intrxn) terms of each age x time ANOVA are shown in A C. *Post different from before exercise (Pre) within group as determined post hoc by Tukey's honest significant difference (HSD) test, p < AU, arbitrary units. 30

41 Figure 2. Effects of an unaccustomed resistance exercise bout on phosphorylation of eukaryotic initiation factor 4E (eif4e) binding protein (4EBP1) (A), eif4e (B), and eif4g (C) 24 h after exercise in young and old subjects. D: representative immunoblots. Bars are means ± SE. Significant main effects of each age x time ANOVA are shown in A C. *Post different from Pre within group as determined post hoc by Tukey's HSD test, p <

42 eukaryotic initiation factors, the 4EBP1 binding partner eif4e was increased in both phosphorylation state (23%, p < 0.001, Figure 2B) and total protein content (19%, p < 0.001). The magnitudes of these changes were similar among young and old subjects. Phosphorylation of the eif4e binding partner, eif4g, was elevated overall 53% (p < 0.001) and this response appeared greater in old subjects despite no significant (p = 0.067) interaction term (Figure 2C). Total eif4g content remained unchanged. Among the signaling targets studied, no age differences were seen at baseline. Upregulated translational signaling 24 h after the unaccustomed resistance loading bout occurred in conjunction with an overall 62% increase in FSR, which was evaluated in a subset of the subjects (n = 14; 8 young, 6 old) (Figure 3). However, unlike the signaling results, increased FSR was driven entirely by young subjects, who experienced Figure 3. Effects of an unaccustomed resistance exercise bout on the fractional synthesis rate (FSR) of mixed muscle protein 24 h after exercise in young and old subjects. Bars are mean ± SE. Results of the age x time ANOVA are shown. *Post different from Pre within group as determined post hoc by Tukey's HSD test, p <

43 a robust 96% increase (p < 0.05) compared with no change among old subjects (p = 0.95). The age x time interaction term did not reach significance (p = 0.084) despite the apparent age difference in responsiveness, owing most likely to the limited sample size in the subset of subjects completing FSR studies. Baseline fasting FSR was nearly identical in the two age groups (young ± 0.006%/h; old ± 0.010%/h). Adaptations during progressive resistance training. Myofiber adaptations are shown in Figure 4. Similar to the acute response signaling results, no age x time interactions were noted for any of the myofiber adaptations to 16 wk of RT. Across all subjects, mean myofiber size increased an average of 1,345 µm 2 (31%) by week 16 (p < 0.001) and this was significant among both young (1,444 µm 2 or 32%, p < 0.005) and old (1,203 µm 2 or 30%, p < 0.05). Hypertrophy was greatest in type II myofibers (P < 0.001) with 38% type II growth on average overall. Within age groups, type II myofiber hypertrophy was seen in both young (1,666 µm 2 or 37%, p < 0.001) and old (1,447 µm 2 or 40%, p < 0.001) (Figure 4A). Type II myofibers were smaller among old subjects (main age effect, p < 0.05), indicative of type II atrophy, a classic phenotypic index of sarcopenia. Type I myofiber size also increased overall (972 µm 2 or 22%, p < 0.001) (data not shown). The type IIx-to-IIa myofiber type shift typical of resistance training was significant within each age group similar to our previous reports (3, 25) (Figure 4B). Including all subjects, the relative distribution of type IIa myofibers increased (p < 0.001) from 48.9% to 61.5%, while the distribution of type IIx myofibers dropped (p < 0.001) from 14.8% to 1.7%. Overall, fewer type IIx fibers were found in old vs. young (main age effect, p < 0.05). As expected, no change in type I myofiber distribution was found. 33

44 Figure 4. Effects of a 16-wk resistance training program on type II myofiber cross-sectional area (CSA) (A) and distribution of type IIa and type IIx myofibers (B) in young and old subjects. Bars are means ± SE. Results of each age x time ANOVA are shown in A and B. *Post different from Pre within group as determined post hoc by Tukey's HSD test, p < Table 1 displays lean mass and strength results. In agreement with myofiber hypertrophy, DEXA-determined thigh lean mass increased overall (p < 0.001) with no significant age x time interaction. Total body lean mass also increased (p < 0.005) and, as expected since the RT program focused on hip and knee extensors, the bulk of this increase was in the thigh compartment. Main time effects (p < 0.001) were noted for all three maximum voluntary dynamic (1RM) strength tests (leg press, knee extension, squat). For example, knee extension 1RM strength (Table 1) rose 44% among young and 38% in old subjects by week 16 (p < 0.001). For all three movements, the majority of the strength gains were acquired by week 8. For example, significant knee extension 1RM strength gains (p < 0.01) were noted in both young (29%) and old (23%) after the first 8 wk of RT. 34

45 Table 1. Descriptive characteristics and effects of resistance training on lean mass and strength in young and old. All Subjects Young Old (n = 36) (n=21) (n=15) Age (yr) 27.9 ± ± 0.9 Height (cm) ± ± 2.8 Weight (kg) 75.4 ± ± 3.9 Lean mass (kg)* Baseline 48.1 ± ± ± weeks 48.8 ± 1.9 A 50.5 ± 2.5 A 46.4 ± 2.8 Thigh lean mass (g)* Baseline 11,612 ± ,341 ± ,591 ± weeks 12,284 ± 565 A 13,040 ± 742 A 11,227 ± 822 A Bilateral knee extension 1RM strength (kg)* Baseline 48.7 ± ± ± weeks 61.9 ± 3.7 A 70.7 ± 4.8 A 49.4 ± 4.3 A 16 weeks 69.2 ± 4.4 A,B 79.2 ± 5.8 A,B 55.1 ± 5.0 A Values are mean ± SE. *Main time effect, p < 0.05; Main age effect, p < 0.05; Age x time interaction, p < 0.05; A Different from baseline within group, p < 0.05; B Different from week 8 within group, p <

46 DISCUSSION Changes in muscle protein synthesis or translation initiation signaling following a single bout of RE or a single exposure to an alternate exogenous stimulus (e.g., insulin or amino acids) are often speculated to be indexes of responsiveness that may predict whether the final desired end point of increased muscle mass would be achieved with repeated exposures. This study is the first to evaluate whether changes in both translational signaling and FSR in response to unaccustomed RE are in agreement with changes in muscle size following long-term RT, and whether aging influences these responses. We report overall that translational signaling was upregulated at multiple control points along the pathway after a single RE exposure, and that modest hypertrophy followed suit as measured after 16 wk of RT. However, contrary to our expectations, age did not influence signaling responses to RE, nor the hypertrophy adaptation to RT. Further, in a subset of participants, we report lack of agreement between FSR and translational signaling responses to RE. Each of these findings is discussed below. With analysis of a putative translational signaling pathway, we found that all of the signaling (phosphorylation) of proteins of interest, except mtor, were upregulated 24 h after unaccustomed RE in this group of 35 subjects. This may seem surprising given the relatively late tissue collection post-re (24 h) certainly we probably missed the peak activation state of some or all targets. However, FSR has been shown to be increased for up to 48 h post-re, and indeed we have found it to be increased at 24 h. Therefore it can be reasonably presumed that at least a subset of proteins that control translation initiation remain active at this time point. Clearly these findings support RE as a relatively potent and long-lasting stimulus of translation initiation. While some have 36

47 found the timeline of phosphorylation of many components of translation initiation pathways, including Akt, mtor, and 4EBP1, to be quite transient (1, 7, 9, 12), increased phosphorylation of Akt and mtor has also been noted as long as h after the final bout of RE 8 wk into a RT program (29). RE-mediated phosphorylation of S6K1 has been shown to correlate with long-term muscle hypertrophy in both rodents (numerous phosphorylation sites combined) (2) and humans (T389 at 30 min postexercise) (43). Additionally, robust phosphorylation of the S6K1 autoinhibitory domain at T421/S424 has been found to occur immediately ( 30 s) after resistance exercise and persist for at least 24 h (10). Full activation of S6K1 requires a series of sequential, differentially regulated phosphorylation steps (11, 37, 46). The autoinhibitory domain regulates access to T389 and therefore must be phosphorylated (S411/S418/T421/S424) first to open the conformation for subsequent T389 phosphorylation. Likewise in sequence, T389 phosphorylation must occur prior to loop site (T229) phosphorylation, T229 being the final activating site required for full kinase activity. T389 is considered the acute response phosphorylation site; thus it is not surprising that T389 phosphorylation occurs within 1 2 h after RE (12, 26) but returns to baseline shortly thereafter (26) and was not detected in our model 24 h after RE. Because the autoinhibitory domain must be phosphorylated first to make T389 accessible, prolonged phosphorylation of this domain would presumably facilitate full kinase activation (T389 followed by T229) with each acute stimulus such as protein feeding. In the present study we found a modest correlation (r = 0.53, p < 0.005) between the percent change in S6K1 autoinhibitory domain phosphorylation (T421/S424) 24 h after the single RE bout and percent change in mean myofiber size after 16 wk of 37

48 RT. Admittedly the zero-order correlation as applied here is an overly simplistic view of a complex series of events required for successful hypertrophy; however, the positive correlation supports the notion that those individuals with prolonged S6K1 autoinhibitory domain phosphorylation following a bout of RE were better poised to respond (with sequential T389 phosphorylation) to other acute anabolic stimuli (e.g., protein feeding) during the time period between bouts of RE. Somewhat surprising to us, we did not find a compelling effect of aging on translational signaling responsiveness to unaccustomed RE. We are aware of only one other human RE study in which such an aging effect was tested after an overnight fast (26). Kumar et al. (26) found increases in S6K1 and 4EBP1 phosphorylation in young (by combining subjects from 3 different exercise intensities) but not old subjects 1 h after RE, with no other differences noted from baseline for up to 4 h after RE. Lack of agreement between our results and those of Kumar likely results from a major difference in study design timing. Kumar detected age differences in responses 1 h postexercise, including the acute response phosphorylation site on S6K1 (T389), which returned to baseline by 2 h post-re. Kumar et al. did not assess phosphorylation of the S6K1 autoinhibitory domain, but based on the sequential activation paradigm its phosphorylation would have been obligatory, at least in the young. That we found no age difference in long-term autoinhibitory domain phosphorylation does not rule out the possibility that aging may affect subsequent T389 phosphorylation in response to acute stimuli. On the other hand, it should not be overlooked that one putative downstream target of fully activated S6K1, RPS6, was robustly phosphorylated in old subjects and, in fact, revealed the only significant age x time interaction found among the signaling targets studied here with a 38

49 greater response in old. Our S6K1 findings generally agree with those of Hornberger et al. (18), who showed using an ex vivo mouse model of passive stretch that S6K1 phosphorylation (both T421/S424 and T389) in response to mechanical strain was not different in extensor digitorum longus muscles isolated from young and old mice. There appeared to be subtle influences of age at different points along the signaling pathway (e.g., within-groups increases in Akt and 4EBP1 phosphorylation only among young and eif4g only among old); however, the predominance of main time effects without age x time interactions in this relatively large data set leads to the general conclusion that translational signaling responsiveness to RE was unaffected by age. Our results regarding myofiber hypertrophy after 16 wk of RT fully support this conclusion. Both age groups achieved significant and comparable myofiber hypertrophy across 16 wk of training. Myofiber hypertrophy is achieved via net protein accretion, a process that may become dependent on the addition of myonuclei if myofiber volume expands substantially due to the likelihood of a threshold or ceiling for the myonuclear domain (31, 32). We have previously shown successful but blunted resistance trainingmediated myofiber hypertrophy among larger cohorts of older adults vs. young (25, 31), with the decreased efficacy among old vs. young attributed largely to failing myonuclear addition (31). In fact, via K-means cluster analysis we reported recently that the population of available satellite cells pretraining, and the propensity to add myonuclei during training, appear to be major factors in determining the magnitude of myofiber hypertrophy achieved during RT (32). In the present study, while the number of myonuclei per fiber in cross sections increased (p < 0.001) from 2.30 to 2.71, the myonuclear domain expanded (p < 0.01) from 1,862 ± 65 µm 2 to 2,087 ± 72 µm 2 per 39

50 nucleus, indicating protein accretion at a rate in excess of myonuclear addition. No age x time interactions were noted, and the posttraining domain size just reached the theoretical threshold of 2,000 µm 2 per nucleus that we have suggested may increase the dependency on myonuclear addition for further myofiber hypertrophy (31). These findings suggest myofiber hypertrophy resulting predominately from protein accretion is not impaired in old, and this is supported by the similar acute translational signaling responses in young and old. Any age-related attenuation of RT-mediated hypertrophy appears to be more closely linked to age effects on satellite cell activity (31), which may not have been a limiting factor in the present study based on the modest myofiber growth achieved. Our finding of increased mixed muscle FSR after unaccustomed RE in young subjects is similar to previous reports (17, 23, 34, 47). Using FSR as the outcome measure, aging has been associated with blunted responsiveness to anabolic stimuli (16, 38, 42), leading to the speculation that repeated exposures would cause impaired adaptation in aging muscle. Sheffield-Moore et al. (42) reported that unlike young, old men were unable to increase FSR 3 h postexercise, a time point previously found to be reflective of FSR at 24 h (44). We too report a failed FSR response to unaccustomed RE in a subset of the older participants but show for the first time that this was not in agreement with the translational signaling results and did not appear to impact the eventual gains in muscle mass and myofiber hypertrophy at the conclusion of a 16-wk RT program. In fact, excluding the 22 subjects lacking FSR assessments, myofiber hypertrophy was similarly achieved (p < 0.05) in the subset of young and old with measured FSR, and in this subset acute response FSR did not correlate with the 40

51 magnitude of eventual hypertrophy. We are not the first to report lack of agreement between acute translational signaling and FSR responses; however, this is the first report of such a phenomenon in response to RE. During intravenous delivery of amino acids and incrementing insulin concentrations, Greenhaff et al. (15) found a dose response for Akt (S473) and S6K1 (T389) phosphorylation while FSR failed to increase with rising insulin delivery. The authors concluded that their data do not support the commonly accepted model by which increases in FSR "follow from proportionate alterations in the activity of signaling molecules"; consequently, Greenhaff and colleagues consider this model an oversimplification (15). We agree with Greenhaff that unknowns such as the temporal relationship between phosphorylation (i.e., signaling) and anabolism (i.e., FSR), or the degree of phosphorylation indicative of true signaling activity, complicate interpretation. Without question such unknowns make data interpretation more difficult; however, based on the fasting FSR vs. fasting signaling responses seen 24 h after unaccustomed RE, our results suggest fasting FSR may not be an ideal index of responsiveness to an unaccustomed stimulus, particularly when the outcome of interest is myofiber growth following several weeks of RT. While the findings reported herein are novel and should prove useful for future studies, we fully appreciate the limitations. First, FSR measurements were limited to a subset of participants. The FSR infusion studies were considered a "substudy," and therefore, although we aimed to recruit 10 young and 10 old subjects into this substudy, we were unable to reach this goal. (Two additional young subjects completed the repeat FSR assessments but did not complete the hypertrophy training program, and no more older subjects volunteered for the substudy.) Second, the assessment of translational 41

52 signaling was certainly not exhaustive and a more thorough analysis of S6K1 downstream mediators may prove revealing. Third, a single postexercise time point may limit data interpretation; however, we have found the 24 h time point to be quite meaningful in this and prior (3, 22, 24, 32) studies. Fourth, we recognize that the power to detect age x time interactions may have been limited by sample size. On the other hand, using similar or in some cases identical procedures and fewer subjects, we have previously found age x time interactions for both myofiber hypertrophy and cell signaling following RT (17 young vs. 13 old) (24), as well as group x time differences in fasting FSR with only five to six subjects per group following unloading (14). In summary, contrary to our hypothesis, translational signaling responses to unaccustomed RE were not blunted in older adults, suggesting the protein synthesis machinery is relatively intact and reasonably responsive among old. Further, young and old experienced similar magnitudes of myofiber hypertrophy, which appeared largely by protein accretion based on myonuclear domain expansion. Further study is warranted to better understand the temporal relationship between translational signaling and rates of muscle protein synthesis, particularly among older adults. 42

53 GRANTS Funding for this work was provided by National Institute on Aging Grants R01- AG (M. M. Bamman) and F30-AG (D. L. Mayhew), a Veterans Affairs Merit Grant (M. M. Bamman), and General Clinical Research Center Grant M01-RR ACKNOWLEDGMENTS We are indebted to the research subjects for invaluable contributions to this work. We thank S. C. Tuggle for administering the resistance training program. 43

54 REFERENCES 1. Atherton PJ, Babraj J, Smith K, Singh J, Rennie MJ Wackerhage H. Selective activation of AMPK-PGC-1alpha or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation. FASEB J 19: , Baar K and Esser K. Phosphorylation of p70(s6k) correlates with increased skeletal muscle mass following resistance exercise. Am J Physiol Cell Physiol 276: C120 C127, Bamman MM, Petrella JK, Kim JS, Mayhew DL Cross JM. Cluster analysis tests the importance of myogenic gene expression during myofiber hypertrophy in humans. J Appl Physiol 102: , Bamman MM, Ragan RC, Kim JS, Cross JM, Hill VJ, Tuggle SC Allman RM. Myogenic protein expression before and after resistance loading in 26- and 64-yrold men and women. J Appl Physiol 97: , Biolo G, Maggi SP, Williams BD, Tipton KD Wolfe RR. Increased rates of muscle protein turnover and amino acid transport after resistance exercise in humans. Am J Physiol Endocrinol Metab 268: E514 E520, Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ Yancopoulos GD. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3: , Bolster DR, Kubica N, Crozier SJ, Williamson DL, Farrell PA, Kimball SR Jefferson LS. Immediate response of mammalian target of rapamycin (mtor)- mediated signalling following acute resistance exercise in rat skeletal muscle. J Physiol 553: , Chesley A, MacDougall JD, Tarnopolsky MA, Atkinson SA Smith K. Changes in human muscle protein synthesis after resistance exercise. J Appl Physiol 73: , Coffey VG, Zhong Z, Shield A, Canny BJ, Chibalin AV, Zierath JR Hawley JA. Early signaling responses to divergent exercise stimuli in skeletal muscle from well-trained humans. FASEB J 20: , Deldicque L, Atherton P, Patel R, Theisen D, Nielens H, Rennie MJ Francaux M. Decrease in Akt/PKB signalling in human skeletal muscle by resistance exercise. Eur J Appl Physiol 104: 57 65,

55 11. Dennis PB, Pullen N, Pearson RB, Kozma SC Thomas G. Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) activation loop phosphorylation. J Biol Chem 273: , Dreyer HC, Fujita S, Cadenas JG, Chinkes DL, Volpi E Rasmussen BB. Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. J Physiol 576: , Evans W, Phinney S Young V. Suction applied to a muscle biopsy maximizes sample size. Med Sci Sports Exerc 14: , Ferrando AA, Tipton KD, Bamman MM Wolfe RR. Resistance exercise maintains skeletal muscle protein synthesis during bed rest. J Appl Physiol 82: , Greenhaff PL, Karagounis LG, Peirce N, Simpson EJ, Hazell M, Layfield R, Wackerhage H, Smith K, Atherton P, Selby A Rennie MJ. Disassociation between the effects of amino acids and insulin on signaling, ubiquitin ligases, and protein turnover in human muscle. Am J Physiol Endocrinol Metab 295: E595 E604, Guillet C, Prod'homme M, Balage M, Gachon P, Giraudet C, Morin L, Grizard J Boirie Y. Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans. FASEB J 18: , Hasten DL, Pak-Loduca J, Obert KA Yarasheski KE. Resistance exercise acutely increases MHC and mixed muscle protein synthesis rates in and yr olds. Am J Physiol Endocrinol Metab 278: E620 E626, Hornberger TA, Mateja RD, Chin ER, Andrews JL Esser KA. Aging does not alter the mechanosensitivity of the p38, p70s6k, and JNK2 signaling pathways in skeletal muscle. J Appl Physiol 98: , Hornberger TA, Sukhija KB, Wang XR Chien S. mtor is the rapamycinsensitive kinase that confers mechanically-induced phosphorylation of the hydrophobic motif site Thr(389) in p70(s6k). FEBS Lett 581: , Iijima Y, Laser M, Shiraishi H, Willey CD, Sundaravadivel B, Xu L, McDermott PJ Kuppuswamy D. c-raf/mek/erk pathway controls protein kinase C- mediated p70s6k activation in adult cardiac muscle cells. J Biol Chem 277: , Kim JS, Kosek DJ, Petrella JK, Cross JM Bamman MM. Resting and loadinduced levels of myogenic gene transcripts differ between older adults with 45

56 demonstrable sarcopenia and young men and women. J Appl Physiol 99: , Kim JS, Petrella JK, Cross JM Bamman MM. Load-mediated downregulation of myostatin mrna is not sufficient to promote myofiber hypertrophy in humans: a cluster analysis. J Appl Physiol 103: , Kim PL, Staron RS Phillips SM. Fasted-state skeletal muscle protein synthesis after resistance exercise is altered with training. J Physiol 568: , Kosek DJ Bamman MM. Modulation of the dystrophin-associated protein complex in response to resistance training in young and older men. J Appl Physiol 104: , Kosek DJ, Kim JS, Petrella JK, Cross JM Bamman MM. Efficacy of 3 days/wk resistance training on myofiber hypertrophy and myogenic mechanisms in young vs. older adults. J Appl Physiol 101: , Kumar V, Selby A, Rankin D, Patel R, Atherton P, Hildebrandt W, Williams J, Smith K, Seynnes O, Hiscock N Rennie MJ. Age-related differences in the doseresponse relationship of muscle protein synthesis to resistance exercise in young and old men. J Physiol 587: , Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, Price SR, Mitch WE Goldberg AL. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J 18: 39 51, Lecker SH, Solomon V, Mitch WE Goldberg AL. Muscle protein breakdown and the critical role of the ubiquitin-proteasome pathway in normal and disease states. J Nutr 129: 227S 237S, Leger B, Cartoni R, Praz M, Lamon S, Deriaz O, Crettenand A, Gobelet C, Rohmer P, Konzelmann M, Luthi F Russell AP. Akt signalling through GSK- 3beta, mtor and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy. J Physiol 576: , Nader GA. Molecular determinants of skeletal muscle mass: getting the "AKT" together. Int J Biochem Cell Biol 37: , Petrella JK, Kim JS, Cross JM, Kosek DJ Bamman MM. Efficacy of myonuclear addition may explain differential myofiber growth among resistance-trained young and older men and women. Am J Physiol Endocrinol Metab 291: E937 E946, Petrella JK, Kim JS, Mayhew DL, Cross JM Bamman MM. Potent myofiber hypertrophy during resistance training in humans is associated with satellite cell- 46

57 mediated myonuclear addition: a cluster analysis. J Appl Physiol 104: , Petrella JK, Kim JS, Tuggle SC, Hall SR Bamman MM. Age differences in knee extension power, contractile velocity, and fatigability. J Appl Physiol 98: , Phillips SM, Parise G, Roy BD, Tipton KD, Wolfe RR Tamopolsky MA. Resistance-training-induced adaptations in skeletal muscle protein turnover in the fed state. Can J Physiol Pharmacol 80: , Phillips SM, Tipton KD, Aarsland A, Wolf SE Wolfe RR. Mixed muscle protein synthesis and breakdown after resistance exercise in humans. Am J Physiol Endocrinol Metab 273: E99 E107, Phillips SM, Tipton KD, Ferrando AA Wolfe RR. Resistance training reduces the acute exercise-induced increase in muscle protein turnover. Am J Physiol Endocrinol Metab 276: E118 E124, Pullen N Thomas G. The modular phosphorylation and activation of p70s6k. FEBS Lett 410: 78 82, Rasmussen BB, Fujita S, Wolfe RR, Mittendorfer B, Roy M, Rowe VL Volpi E. Insulin resistance of muscle protein metabolism in aging. FASEB J 20: , Rennie MJ, Wackerhage H, Spangenburg EE Booth FW. Control of the size of the human muscle mass. Annu Rev Physiol 66: , Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, Stitt TN, Yancopoulos GD Glass DJ. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol 3: , Shanely RA, Zergeroglu MA, Lennon SL, Sugiura T, Yimlamai T, Enns D, Belcastro A Powers SK. Mechanical ventilation-induced diaphragmatic atrophy is associated with oxidative injury and increased proteolytic activity. Am J Respir Crit Care Med 166: , Sheffield-Moore M, Paddon-Jones D, Sanford AP, Rosenblatt JI, Matlock AG, Cree MG Wolfe RR. Mixed muscle and hepatic derived plasma protein metabolism is differentially regulated in older and younger men following resistance exercise. Am J Physiol Endocrinol Metab 288: E922 E929, Terzis G, Georgiadis G, Stratakos G, Vogiatzis I, Kavouras S, Manta P, Mascher H Blomstrand E. Resistance exercise-induced increase in muscle mass correlates 47

58 with p70s6 kinase phosphorylation in human subjects. Eur J Appl Physiol 102: , Tipton KD, Borsheim E, Wolf SE, Sanford AP Wolfe RR. Acute response of net muscle protein balance reflects 24-h balance after exercise and amino acid ingestion. Am J Physiol Endocrinol Metab 284: E76 E89, Tokuda H, Hatakeyama D, Shibata T, Akamatsu S, Oiso Y Kozawa O. p38 MAP kinase regulates BMP-4-stimulated VEGF synthesis via p70 S6 kinase in osteoblasts. Am J Physiol Endocrinol Metab 284: E1202 E1209, Weng QP, Andrabi K, Kozlowski MT, Grove JR Avruch J. Multiple independent inputs are required for activation of the p70 S6 kinase. Mol Cell Biol 15: , Yarasheski K, Zachwieja J Bier D. Acute effects of resistance exercise on muscle protein synthesis rate in young and elderly men and women. Am J Physiol Endocrinol Metab 265: E210 E214, Yarasheski KE, Zachwieja JJ Bier DM. Acute effects of resistance exercise on muscle protein synthesis rate in young and elderly men and women. Am J Physiol Endocrinol Metab 265: E210 E214,

59 eif2bε INDUCES CAP-DEPENDENT TRANSLATION AND SKELETAL MUSCLE HYPERTROPHY by DAVID L. MAYHEW, TROY A. HORNBERGER, HANNAH C. LINCOLN, AND MARCAS M. BAMMAN In preparation for Journal of Biological Chemistry Format adapted for dissertation

60 ABSTRACT The purpose of this study was to resolve which signaling components known to control mrna translation initiation in skeletal muscle are responsive to mechanical load and may be largely responsible for myofiber hypertrophy. To accomplish this, we first utilized a human cluster model in which skeletal muscle samples from subjects with widely divergent hypertrophic responses to resistance training were used for the identification of signaling proteins associated with the degree myofiber hypertrophy. We found that of 11 translational signaling molecules examined, the response of p70s6k (T421/S424) phosphorylation and total eif2bε protein abundance after a single bout of unaccustomed resistance exercise was associated with myofiber hypertrophy following 16 wks of training. Follow up studies revealed that overexpression of eif2bε alone was sufficient to induce an 87% increase in cap-dependent translation in L6 myoblasts in vitro and 21% hypertrophy of myofibers in mouse skeletal muscle in vivo. However, genetically altering p70s6k activity had no impact on eif2bε protein abundance in mouse skeletal muscle in vivo or multiple cell lines in vitro, suggesting that the two phenomena were not directly related. These are the first data that mechanistically link eif2bε abundance to skeletal myofiber hypertrophy, and indicate that both eif2bε abundance and p70s6k phosphorylation may at least partially underlie the widely divergent hypertrophic phenotypes in human muscle exposed to mechanical stimuli. 50

61 INTRODUCTION Translation initiation pathways, including PI3K/Akt/mTOR, PI3K/Akt/GSK3, and eif2b are key determinates of cell size regulation in a number of model systems, including skeletal muscle (9, 25, 34, 48). Furthermore, mechanical load, a stimulus that induces muscle cell (i.e., myofiber) hypertrophy, increases the activity of translation initiation pathways in both animal (4, 42) and human muscle (8, 14, 16, 35) leading to increased functional and metabolic reserves of the tissue. It is therefore important to identify which endogenous proteins controlling mrna translation initiation are most influential in determining myofiber hypertrophy in response to mechanical load. Identification of these in vivo regulatory mechanisms will not only reveal basic physiological function but will also provide targets that, when altered therapeutically, may more closely mimic the in vivo hypertrophic response to mechanical load. Translation initiation is thought to be the rate limiting step in protein synthesis (3). An extensively studied signaling pathway that has been shown to increase translation initiation rates of 5 -methylguanosine capped mrna is that involving the serine/threonine kinase mammalian target of rapamycin (mtor). As a component of the mtor complex 1 (mtorc1), mtor phosphorylates at least two downstream targets that control translation: eif4e binding protein 1 (4E-BP1) and the 70 kda isoform of the ribosomal protein S6 kinase (p70s6k). 4E-BP1 binds the cytosolic mrna cap binding protein, eif4e, which precludes eif4e binding to eif4g and prevents formation of the eif4f complex. mtorc1-mediated phosphorylation of 4E-BP1 relieves this inhibition, allowing eif4f formation. The specific mechanisms of p70s6k-induced translation initiation are not well defined, but may involve phosphorylation of rps6, eif4b, mtor, 51

62 or SKAR (27, 46, 47, 49). Together, mtorc1-dependent signaling aids in the creation of a mature, translation-competent ribonucleoprotein complex. Another component of the translational machinery that exerts rate-limiting influence over overall translation initiation in some scenarios is eif2 (31). This initiation factor, when bound to GTP, complexes with methionine trna (Met i -trna) to form the ternary complex. With the aid of a translation-competent ribonucleoprotein complex, the formation of which is regulated at least in part by mtorc1 activity (see above), the ternary complex recognizes the AUG start codon, thereby supplying the initial amino acid in a nascent polypeptide chain. When AUG codon recognition occurs, GTP is hydrolyzed to GDP, which induces dissociation of multiple proteins from the mrna, including eif2 GDP. Since the affinity of eif2 for GDP is much higher than for GTP, GDP dissociation must be catalyzed in order to recycle eif2 to the GTP bound form (40); this process is performed by eif2b, which is composed of α-ε subunits (56). Importantly, the total abundance of the catalytic ε subunit was shown to be regulated by mechanical load in skeletal muscle in vivo at the level of mrna translation (32). The mrna and protein abundance of the other (α-γ) subunits was not affected by mechanical load (32), while exogenous overexpression of eif2bε was sufficient to increase overall eif2b guanine nucleotide exchange factor (GEF) activity (54) and overall protein synthesis (5, 25). Therefore, of the components of the eif2b holoenzyme complex, eif2bε appears to be rate limiting for eif2b GEF activity and overall protein synthesis (at least under some circumstances), and is specifically translated in response to mechanical stimuli in skeletal muscle. However, the specific contribution of increased eif2bε abundance to the overall phenotypic outcome of myofiber hypertrophy remains obscure. 52

63 Others have utilized the protein synthetic and muscle cell signaling response to an acute bout of unaccustomed resistance exercise as synonymous with myofiber hypertrophy after a period of prolonged training in combined subject cohorts, and that the specific signaling proteins altered by mechanical load are implicit drivers of myofiber hypertrophy over time (11, 15, 59). However, the multifaceted nature of the signaling responses of mechanically loaded skeletal muscle make it particularly difficult to segregate mechanisms that are instructive for, as opposed to simply permissive of or coincident with, myofiber hypertrophy in vivo. Furthermore, although specific signaling proteins have been identified as mediators of cell size in other model systems, it does not necessitate these proteins as rate limiting in mechanically loaded human muscle. We have previously applied K-means cluster analysis to delineate those who experience differing degrees of hypertrophy in response to a standardized lower extremity resistance training program, thereby allowing some degree of dissection of the mechanisms underlying distinct hypertrophic phenotypes in humans (6, 29, 44). Using this model the potential requirements of specific signaling proteins for load-mediated hypertrophy can be studied by determining which factors are differentially regulated in those individuals who experience robust (extreme responders, XR), moderate (moderate responder, MR), or no hypertrophy (non-responders, NR) despite normal fiber type shifting in response to the same relative stimulus (6). Thus, NR have a specific deficit in the ability to hypertrophy in response to mechanical load. Given the limitations of human research, this clustercentered model may be the most robust analytical approach available to reveal the regulatory mechanisms mediating mechanical load-induced hypertrophy as opposed to those induced by mechanical load per se but not instructive for hypertrophy. 53

64 The purpose of this study was to resolve which signaling components known to control translation initiation in skeletal muscle are responsive to mechanical load and may be largely responsible for myofiber hypertrophy in vivo. To accomplish this, we employed a multilayered approach beginning with a human cluster model for initial identification of potential rate limiters, followed by targeted studies in a mouse model and in vitro approaches to confirm whether signaling proteins identified in the human observational study were sufficient to induce myofiber hypertrophy. We found that of 11 translational signaling molecules examined in humans, the response of p70s6k (T421/S424) phosphorylation and eif2bε protein abundance 24 h after a single bout of unaccustomed resistance exercise was coincident with myofiber hypertrophy following 16 wk of resistance training. Phosphorylation and/or total content of other signaling components upstream of the translational apparatus were not associated with the degree of myofiber hypertrophy in this model. Follow-up studies therefore focused on eif2bε and p70s6k. Overexpression of eif2bε alone was sufficient to induce an 87% increase in cap-dependent translation in L6 myoblasts in vitro and 21% hypertrophy in mouse skeletal muscle in vivo. However, we found that genetically altering p70s6k activity had no influence over eif2bε protein abundance in mouse skeletal muscle in vivo or multiple cell lines in vitro. These are the first data that mechanistically link eif2bε abundance to skeletal myofiber hypertrophy, and that this may at least partially underlie the widely divergent hypertrophic phenotypes in human muscle exposed to mechanical stimuli. 54

65 METHODS Plasmid construction. Plasmid DNA containing mouse eif2bε (MGC IMAGE clone ID ) was purchased from Invitrogen (Carlsbad, CA). Both the full-length and C-terminal deletion mutant (truncated after amino acid 529) were subcloned, each with the addition of a C-terminal HA-tag, into the prk7 vector using SalI/EcoRI sites. GFP was cloned into the same vector using identical sites. Myc-tagged p70s6k mutants (D3E-T389E and D3E-T389A) were kindly provided by Dr. George Thomas (University of Cincinnati) and have been described previously (43). The pδemcv luciferase reporter (12) was a gift from Dr. Sunnie Thompson (University of Alabama at Birmingham). The plasmids encoding GFP, HA-Rheb, and GST-tagged p70s6k used in mouse overexpression studies have been previously described (23). Human subjects. Sixty-six adults recruited from the Birmingham, Alabama metropolitan area completed the 16 wk resistance training study. All participants were screened by health history questionnaire and older subjects also passed a physical exam and graded exercise stress test. Subjects were not obese (BMI<30) and free of any musculoskeletal or other disorders that might have affected their ability to complete testing and/or resistance training. Subjects had not experienced any leg resistance training in the five years prior to initiating the study protocol. None of the subjects were being treated with exogenous testosterone or other pharmacologic interventions known to influence muscle mass or that may have interacted with the exercise stimulus (such as GH, IGF-1, or immunosuppressive therapy). The project was approved by the Institutional Review Boards of both the University of Alabama at Birmingham and the 55

66 Birmingham Veterans Affairs Medical Center. Written informed consent was obtained from each volunteer prior to participation. Using statistical (K-means) cluster analysis, subjects were classified post hoc into three clusters based on changes in vastus lateralis mean myofiber cross sectional area (MFA, μm 2 ) across 16 wk training as described previously (6). The resulting three clusters XR (n=17; +2,475 ± 140 µm 2 MFA), MR (n=32; +1,111 ± 46 µm 2 ), and NR (n=17; 16 ± 99 µm 2 ) were characterized in detail elsewhere (6). It is noteworthy for this report that the clusters did not differ in training intensity, volume, or weekly adherence to the program. A random subset of subjects (n =10 XR, 16 MR, and 8 NR) was analyzed for translation initiation markers in the current study. Resistance training. A more extensive description of the resistance training program performed by these subjects is provided elsewhere (6). Briefly, participants completed a 16 wk training regimen consisting of 3 sets of 8-12 repetitions at 80% of their individual 1-repetition maximum (1-RM) performance with 90 seconds recovery between sets on the free-weight squat, machine leg press, and knee extension exercises performed 3 times per week. All participants were supervised by a Certified Strength and Conditioning Specialist (National Strength and Conditioning Association) or Certified Health Fitness Instructor (American College of Sports Medicine). Human tissue collection and processing. Fasted morning muscle biopsy samples were collected in the GCRC using routine methods (19) at baseline, 24 h after the first bout of resistance exercise at wk 1, and 24 h after a bout of resistance exercise at wk 16 of the training program. Approximately 70 mg of tissue was mounted cross-sectionally in 56

67 liquid nitrogen-cooled isopentane for subsequent histological analysis and the remainder was weighed, divided, and snap frozen (30-35 mg per tube) in liquid nitrogen. Snap frozen samples were separated into cytosolic and membrane fractions as described previously (35), with Western blotting performed on the cytosolic fraction only. We chose to sample at 24 h post-exercise since muscle protein synthesis has been found to be elevated at this time point (45), and indeed we have found this to be the case in a subset of the subjects examined here (35). Although many of the transient cell signaling events have returned to baseline levels by 24 h, we postulated that the 24 h time point would allow us to more easily witness events that drive protein synthesis during recovery without interference from the myriad of processes activated during and shortly after resistance exercise. Animals. All animal experiments in this study followed protocols approved by the Animal Care and Use Committee at the University of Wisconsin - Madison. Female FVB mice of 8-10 weeks age were obtained from Jackson Laboratories and randomly assigned to experimental treatments. Skeletal muscle transfection (electroporation). Mice were anesthetized with 100 mg/kg ketamine plus 10 mg/kg xylazine and a small incision was made through the skin covering the TA muscle. A 27-gauge needle was used to inject plasmid DNA solution (2.5 μg/μl for GFP, 4.0 μg/μl for eif2bε-ha and eif2bε-s535a-ha, dissolved in sterile PBS) into the proximal (6 μl) and distal (6 μl) ends of the muscle belly. Similar methods were used for injection of GFP, HA-Rheb, and GST-p70S6K, and are described elsewhere (23). Following the injections, electric pulses were applied through two stainless steel pin electrodes (1 cm gap, Harvard Apparatus) laid on top of the proximal 57

68 and distal myotendinous junctions. Eight 20 ms square-wave electric pulses at a frequency of 1 Hz were delivered with an ECM 830 electroporation unit (BTX) at a field a strength of 170 V/cm. Following the electroporation procedure, the incision was closed with Vetbond surgical glue (Henry Schein, Melville, NY) and the mice were allowed to recover for 2 or 7 days, depending upon the experiment. Measurements of transfected muscle fiber cross sectional area. Muscles were excised and fixed in ice cold PBS containing 4% paraformaldehyde with gentle rocking at 4ºC for 30 min. The fixed muscles were submerged in optimal cutting temperature compound and frozen in liquid nitrogen chilled isopentane. Cross sections (10 µm thick) from the mid-belly of the muscle were obtained with a cryostat and fixed in -20 C acetone for 10 min. Sections were warmed to room temperature for 5 min and then rehydrated with cool steam vapors. Under gentle rocking, the rehydrated sections were incubated in PBS for 15 min followed by a 20min incubation in solution A (PBS containing 0.5% bovine serum albumin (BSA) and 0.5% Triton X-100). Sections were then incubated with rabbit anti-laminin (Sigma-Aldrich, St. Louis, MO) and rat anti-ha (Roche, Madison, WI) antibodies dissolved in solution A for 1 h at room temperature. Sections were washed with PBS and then incubated with anti-rabbit TRITC-conjugated and anti-rat FITC conjugated secondary antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA) dissolved in solution A for 1 h at room temperature. Finally, the sections were washed with PBS and mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA). Transfected fibers (HA or GFP positive) and laminin were identified in dual fluorescent images captured through FITC and TRITC cubes on a Nikon 80i epifluorescence microscope. The images were merged with Nikon NIS Elements D image 58

69 analysis software and the cross-sectional area (CSA) of randomly selected transfected and non-transfected fibers per sample were measured by tracing the laminin stain of individual fibers. All CSA analyses were performed by investigators blinded to the treatment. The CSA of the transfected and non-transfected fibers were plotted on a histogram and the average CSA for each fiber type per sample was calculated. Cell culture. C2C12, L6 (ATCC, Manassas, VA), and HEK293T cells (gift from Dr. Sunnie Thompson, University of Alabama at Birmingham) were propagated in DMEM supplemented with 10% FBS, 2 mm Glutamax (Invitrogen), and 100 U/ml penicillin/streptomycin. All cells were grown in a 37 O C humidified incubator at 5% CO 2. Transient transfections. C2C12 and L6 cells were grown to 80-90% confluence on 6 well plastic plates. Cells were trypsinized, replated onto collagen-i (6.7 μg/cm 2 ) coated plates, and immediately transfected with 2 μg of either constitutively active (D 3 E- T389E) or dominant negative (D 3 E-T389A) myc-tagged p70s6k, or prk5 empty vector control using Lipofectamine 2000 (Invitrogen) per the manufacturer s instructions. HEK293T cells were transfected while adherent. The day following transfection C2C12 and HEK293T were exposed to serum-free media for 24 h prior to harvest, while L6 cells were exposed to 0.5% FBS-containing media (complete serum starvation resulted in L6 cell death in our hands). Cells were harvested in modified RIPA buffer (50 mm TRIS- HCl ph 7.4, 150 mm NaCl, 1 mm EDTA, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and 1:100 each Sigma P2714, P2850, and P5726), and homogenized with repeated pipette tip flushing. Lysates were centrifuged at 5000xg for 5 min and equal protein loads of the soluble fraction, as determined by the BCA protein assay, were subjected to Western blotting as described below. Luciferase reporter experiments were carried out on 59

70 12 well plates of L6 myoblasts transfected with 20 ng of pδemcv luciferase reporter and 800 ng of either wild type mouse eif2bε-ha, eif2bε-δc-ha, or HA-GFP. Twentyfour h after transfection cells were cultured in 0.5% FBS-containing media for an additional 24 h before determination of Renilla luciferase activity. All experiments were carried out in quadruplet. Immunoblotting. Immunoblotting was performed on lysates using established methods in our laboratory (7). Samples were run on 4-15% Tris-HCl SDS-PAGE (BioRad) or 4-12% Bis-Tris SDS-PAGE (Invitrogen) gel matrices with 35 µg (in vivo human and mouse lysates) or 20 µg (in vitro cell lysates) total protein loaded into each well and transferred to PVDF membranes. Primary antibodies used included the p85 subunit of PI3K (Upstate), total eif2bε, p(s473)- and total Akt, p(s2448)- and total mtor, p(t421/s424)-, p(t389)-, and total p70s6k, p(s9)- and total GSK3β, p(s21)- GSK3α, p(t37/46)- and total 4E-BP1, p(s209)- and total eif4e, p(s1108)- and total eif4g, p(s240/244)- and total rps6, myc-tag, rabbit HA-tag, p(t197/202)- (Cell Signaling Technologies) and total (Santa Cruz) Mnk1, and rat HA-tag (Roche). Ideal primary antibody concentrations were determined in preliminary experiments and were 1:2000 (v/v) for p85 PI3K, 1:2500 for p-gsk3α/β, GSK3β, p(s240/244)-rps6, rps6, and p70s6k, 1:3000 for α-tubulin, myc-tag, and rabbit HA-tag, and 1:1000 for all other antibodies. HRP-conjugated secondary antibody was used at 1:50,000 (w/v) followed by chemiluminescent detection in a BioRad ChemiDoc imaging system with band densitometry performed using BioRad Quantity One (software package 4.5.1). Parameters for image development in the ChemiDoc were consistent across all membranes using predefined saturation criteria for the CCD camera. 60

71 Luciferase activity. Transfected L6 cells plated on 12 well plates were lysed in 250 μl Passive Lysis Buffer (Promega). Renilla luciferase activity was determined with the Dual Luciferase Reporter assay kit (Promega) using a Berthold Lumat LB9507 luminometer. Statistics. Translation initiation signaling was assessed for most proteins in n = 10 XR, 16 MR, and 8 NR. Due to sample availability, some proteins were analyzed with a slightly smaller sample size. 3 x 3 (response cluster x time) repeated measures ANOVA were performed for each protein to determine differences in total protein expression levels and phosphorylation state across time. Tukey HSD tests were performed post hoc to localize main effects, while Fisher LSD was used to localize interaction effects. Data for C2C12, L6, and HEK293T cells were subjected to ANOVA and, when appropriate, Tukey HSD performed post hoc. For all statistics, significance was set at the p = 0.05 level. 61

72 RESULTS Translation initiation signaling proteins not differentially regulated by human response cluster. We have previously reported on three distinct phenotypic groups of humans that differ in their hypertrophic responsiveness to 16 wk resistance training (6). Further, it is established that mechanical load-induced myofiber hypertrophy is regulated in large part at the level of mrna translation initiation (3, 30). We thus chose to investigate which signaling components that control translation initiation were differentially regulated among response clusters and may account for the observed phenotypic disparities. Repeated measures ANOVA revealed main time effects (p < 0.05) for p(s473)- Akt, which increased 51% at wk 1 (all % increases are with respect to baseline unless otherwise noted), and total Akt, which increased 37% at wk 16 (Figure 1). These changes were noted without any change in the total content of the p85 regulatory subunit of PI3K, an upstream regulator of Akt (Figure 1). GSK3 is a known downstream target of Akt and negative regulator of eif2bε catalytic activity (10). Phosphorylation of the α isoform of this protein increased 15% at wk 16, however it did so equally in all response clusters (Figure 1). Despite the increase in p(s473)-akt at wk 1, neither an mtor phosphorylation site downstream of Akt, S2448, nor total mtor levels were altered at any time point (Figure 1). Downstream of mtor, the phosphorylation state of the eif4e binding protein, 4E-BP1, trended toward significance (p = 0.051), with a 23% increase occurring from baseline to wk 1, while the total expression level of 4E-BP1 was significantly decreased by 25% at wk 16 (p < 0.05) (Figure 1). Thus, while the levels of p(s473)-akt, total Akt, p(s21)-gsk3α, and total 4E-BP1 were altered significantly by 62

73 A. A.U Baseline 24 h after first bout 24 h after final bout B. A.U Time: p < Time: p < * * * C PI3K(p85) D p(s473)-akt Akt Time: p = Time: p < A.U A.U * E. A.U p(s2448)-mtor mtor p(t37/46)-4ebp1 Time: p < Time: p < A.U. 200 Time: p < * * 160 * * F * 4EBP p(s209)-eif4e eif4e 60 p(s1108)-eif4g eif4g Figure 1. Resistance exercise-induced changes in translational signaling components not altered differentially by hypertrophy response cluster. Human subjects completed a 16 wk resistance training study as described in the text, with skeletal muscle biopsies taken at baseline and 24 h after the first and last loading bouts. Western blotting was used to determine the phosphorylation and/or abundance of each protein. Since the data were not different among response clusters (non-significant response cluster x time interaction term), the data are presented collapsed across all subjects. *, Different from baseline, p < 0.05;, Different from first bout response, p < Values are mean ± SEM arbitrary units based on optical density of immunoblotted protein bands, relative to baseline levels within each cluster, n=

74 mechanical load, these alterations occurred to an equal degree in XR, MR, and NR and could therefore not explain the differences in hypertrophy noted among these groups. The phosphorylation of eif4e on S209 increased 22% at wk 1 in all clusters (p < 0.05) and returned to baseline levels by wk 16 (Figure 1). Total protein expression of eif4e increased 18% at wk 1 and remained 14% elevated at wk 16 (p < 0.05). eif4g, a protein that complexes with eif4e and eif4a to form eif4f (55), associates with both the cytosolic and membrane fractionated compartments in our hands; it is important to note that we report only the cytosolic component here. Ser1108 phosphorylation of cytosolic eif4g increased 52% at wk 1 and 39% at wk 16 (p < 0.05) without any change in the total cytosolic expression level (p > 0.05) (Figure 1). Therefore the regulation of initiation factors 4E and 4G, while being responsive to mechanical load, were equally responsive in all response clusters and could not predict differential myofiber hypertrophy among clusters. Translation initiation signaling proteins differentially regulated by human response cluster. Another downstream effector of mtor, p70s6k, is regulated by phosphorylation at numerous sites including T421/S424 in the autoinhibitory domain, and the putative mtor-specific phosphorylation site T389 in the linker domain (24, 43). Immunoblotting human skeletal muscle lysate revealed that phosphorylation of T421/S424 was increased by 193% in XR at wk 1 (p < 0.05) and returned to baseline by wk 16, while in NR a 164% increase was not evident until wk 16 (p < 0.05, Figure 2). MR appeared to be a mixture of the two responses, which may have diluted the effect at any one time point (p > 0.05). These observations occurred without a significant change 64

75 p(s9)-gsk3β (A.U.) p(t197/202)-mnk1 (A.U.) eif2bε (A.U.) p(t421/s424)-p70s6k (A.U.) Figure 2. Resistance exercise-induced changes translational signaling components that were altered differentially by hypertrophy response cluster. Human subjects completed a 16 wk resistance training study as described in the text, with skeletal muscle biopsies taken at baseline and 24 h after the first and last loading bouts. Western blotting was used to determine the phosphorylation and/or abundance of each protein. Each variable presented here demonstrated a significant response cluster x time interaction term, in contrast to the data presented in Figure 1, and were thus expressed by cluster. Values are mean ± SEM arbitrary units based on optical density of immunoblotted protein bands, relative to baseline levels within each cluster, n=8-10 XR, MR, 8 NR. *, Different from baseline within cluster, p < 0.05;, Different from first bout response within cluster, p <

76 in the expression level of total p70s6k protein in any group (p > 0.05, data not shown). Therefore, the response of T421/S424 phosphorylation was able to effectively distinguish the three response clusters. This suggests that either activation of p70s6k signaling or a kinase responsible for T421/S424 phosphorylation early in a resistance training program may partially underlie the hypertrophic phenotype seen in XR after 16 wk training. eif2bε is the catalytic component of the eif2b holoenzyme, and its activity is required to exchange GDP for GTP on eif2, thereby allowing Met i -trna binding to the AUG start codon to initiate translation. Total eif2bε protein increased 45% and 44% at wk 1 in XR and MR (p < 0.05), respectively, with XR fully, and MR partially returning to baseline levels by wk 16 (Figure 2). While eif2bε expression in NR did not significantly change at any time point, a trend toward an increase from baseline to wk 16 was noted (p = 0.09). Therefore, of the initiation factors studied here, eif2bε was regulated differentially among response clusters, while eif4e and eif4g were regulated similarly among all clusters. Mnk1 has been shown to phosphorylate eif4e and regulate eif4e activity during stressed conditions (13). A significant time x cluster interaction revealed that p(t197/202)-mnk1 increased 95% at wk 16 with respect to wk 1 in NR only (Figure 2). Total Mnk1 expression tended to increase 39% at 16 wk in all clusters combined (p = 0.051, data not shown). The regulation of GSK3β seemed to occur by a different mechanism than the α isoform. Phosphorylation (inhibition) of GSK3β at S9 showed a significant cluster x time interaction; it increased slightly (17%), although significantly, at both wk 1 and wk 16 in 66

77 MR (p < 0.05), and decreased 28% at wk 16 with respect to wk 1 in XR only (p < 0.05, Figure 2). The expression level of total GSK3β was not altered at any time point (p > 0.05, data not shown). A clear trend between GSK3β phosphorylation and hypertrophy was not evident. Therefore, while GSK3β-dependent signaling may have been slightly altered by response cluster across time, the degree to which this modest change may have been physiologically relevant remains to be seen at this time. Since protein, and particularly leucine, ingestion has been shown to stimulate mtorc1-dependent translation initiation irrespective of mechanical load (2, 37), we previously performed dietary analysis on all human subjects, and found that all response clusters had similar intakes of total carbohydrate, total fat, total protein, and leucine (52). Importantly, this finding likely ruled out total protein or leucine intake as a causal mechanism for the differential hypertrophy and translation initiation signaling between response clusters. eif2bε overexpression increased in vitro translation and in vivo myofiber hypertrophy. Since we observed a mechanical load-induced increase in eif2bε among only those subjects who experienced subsequent myofiber hypertrophy (XR and MR), we sought to determine if increased eif2bε was sufficient to induce this response. We first transiently transfected L6 myoblasts with a Renilla luciferase reporter and either GFP, wild type eif2bε, or an eif2bε mutant lacking the C-terminal catalytic domain (eif2bε- ΔC). The translation of the luciferase reporter is driven exclusively by a cap-dependent mechanism, and this technique has been shown previously to be reflective of overall protein synthesis rates (5). Overexpression of eif2bε resulted in an 87% increase in capdependent translation relative to GFP transfected controls (p < 0.05, Figure 3), which is 67

78 Cap-Dependent Translation * GFP 2Bε 2Bε-ΔC Figure 3. eif2bε activity is sufficient to increase cap-dependent translation in vitro. L6 myoblast cells were transfected with a CMV-driven Renilla luciferase reporter and either GFP, wild type eif2bε (2Bε), or a mutant lacking the C-terminal catalytic domain (2Bε-ΔC). Twenty-four h after transfection cells were cultured in 0.5% FBS for an additional 24 h, harvested, and assayed for Renilla luciferase activity expressed relative to GFP control. Values are mean ± SEM, n=4. *, p < 0.05 from GFP control. similar to what has been found previously in other cell types (5). This response was dependent upon the catalytic activity of the protein, as a mutant lacking the catalytic domain failed to alter cap-dependent translation rates from control levels. Thus, increased eif2bε catalytic activity was sufficient to increase overall cap-dependent translation rates in L6 myoblasts in vitro. We wished to determine if the increased cap-dependent translation rates induced by eif2bε overexpression would result in myofiber hypertrophy in the intact animal. Overexpression of eif2bε via electroporation of plasmid DNA into mouse tibialis anterior muscle resulted in a 20% increase in cross-sectional area of transfected fibers 7 d after electroporation (p < 0.05), while expression of GFP had no effect relative to non- 68

79 A. B. GFP Laminin C. D. 2Bε Laminin E. F. 2Bε-S535A Laminin G. * * GFP 2Bε 2Bε- S535A Figure 4. Overexpression of eif2bε or eif2bε-s535a induce skeletal muscle fiber hypertrophy. Mouse TA muscles were transfected with GFP (A-B), eif2bε (2Bε) (C- D), or constitutively active eif2bε (2Bε-S535A) (E-F). At 7 days post transfection, the muscles were collected and cross-sections from the mid-belly of the muscle were subjected to immunohistochemistry for (A) GFP and laminin, (C) 2Bε and laminin, or (E) 2Bε-S535A and laminin. The CSA of transfected fibers (green bars) and nontransfected fibers (black bars) from (B) GFP, (D) 2Bε, or (F) 2Bε-S535A transfected muscles was determined and plotted on a histogram (n = transfected and non-transfected fibers / group). (H) Average CSA of the transfected (+) and nontransfected (-) fibers in GFP, 2Bε and S535A transfected muscles. Values are mean ± SEM, n=5-7 muscles / group. * Significantly different from the non-transfected fibers within a given condition. 69

80 transfected fibers (Figure 4). An eif2bε mutant (S535A) that has previously been reported to act in a constitutively active manner in cardiac myocytes (25) had an effect equal to that of the wild type protein (p < 0.05 vs. non-transfected fibers). The mutated residue is a phosphorylation target of GSK3, which could indicate that GSK3-dependent inhibition of eif2bε may play less of a role in skeletal muscle than in cardiac muscle, or that overexpressed wild type eif2bε may have been present in a quantity too great to be inhibited by endogenous levels of GSK3. In either case, the degree of overexpression of either wild type or the S535A mutant was evidently sufficient to saturate the cellular need for eif2b GEF activity, since we saw equal hypertrophy when either was overexpressed. These data demonstrate that increased abundance of eif2bε was sufficient to induce significant myofiber hypertrophy in mouse skeletal muscle in a short time (7 d), making it likely that the hypertrophy seen in XR and MR humans over 16 wk was at least partially due to augmented protein synthesis secondary to increased abundance of eif2bε. p70s6k signaling did not induce increased eif2bε protein abundance in vitro or in vivo. It is known that p70s6k signaling is necessary for proper regulation of cell size (22, 26, 38); however, the downstream effectors that mediate this process are not well defined. We observed increases in both p70s6k (T421/S424) phosphorylation and eif2bε abundance among XR (Figure 2). Furthermore, Kubica et al. (33) found that mtorc1 signaling was sufficient to induce increased translation of eif2bε, which was blocked by rapamycin administration. Together these data suggested that p70s6k may be the rapamycin-sensitive component downstream of mtorc1 that mediates this 70

81 A. C2C12 L6 HEK293T prk5 p70s6k-d 3 E-T389E p70s6k-d 3 E-T389A eif2bε p70s6k myc-tag p(s240/244)-rps6 rps6 α-tubulin B. C2C12 L6 HEK293T eif2bε (A.U.) prk5 p70s6k-d 3 E-T389E p70s6k-d 3 E-T389A Figure 5. p70s6k signaling does not alter total eif2bε cellular abundance in vitro. (A) C2C12, L6, and HEK293T cells were transfected with wild type, constitutively active (D 3 E-T389E) or dominant negative (D 3 E-T389A) p70s6k, or empty vector control (prk5). Twenty-four hours later cells were exposed to serum free (C2C12 and HEK293T) or 0.5% FBS media (L6) for an additional 24 h, harvested, and analyzed by Western blotting with the indicated antibodies. (B) Values are mean ± SEM for the eif2bε data shown in A, n=4. 71

82 response. In order to test this hypothesis, we transfected multiple cell lines with constitutively active (D 3 E-T389E) or dominant negative (D 3 E-T389A) p70s6k in order to determine the effects of p70s6k signaling on eif2bε protein abundance. In C2C12 mouse myoblasts, alterations in p70s6k activity failed to exert an effect on eif2bε protein abundance (p > 0.05) despite the expected effects on rps6 phosphorylation (a consequence of p70s6k signaling) relative to prk5-transfected control cells (Figure 5). Similar results were obtained in L6 myoblasts. Since C2C12 and L6 myoblasts have a propensity to differentiate under serum withdrawal, we also performed these experiments in the non-myogenic HEK293T cells. Although we obtained nearly 100% transfection efficiency and greater subsequent effects on rps6 phosphorylation in HEK293T cells, there remained no effect on total eif2bε abundance (p > 0.05, Figure 5). This lack of effect occurred despite the ability of each of these cell lines to increase eif2bε in response to other stimuli in our hands (data not shown), indicating that eif2bε levels are in fact malleable in these cell lines. Thus, in 3 different cell lines of myogenic and nonmyogenic origins p70s6k activity had no influence over the cellular steady state abundance, and therefore presumably the synthesis, of eif2bε. Although we found no change in eif2bε abundance with altered p70s6k activity in vitro, the possibility existed that the behavior of the cell lines chosen did not reflect the in vivo condition. Therefore, we chose to investigate whether increased p70s6k activity was sufficient to induce an increase in eif2bε abundance in mouse skeletal muscle in vivo. To do this, we overexpressed wild type p70s6k with or without Rheb, an upstream activator of mtorc1 (60), via electroporation of plasmid DNA into mouse tibialis anterior muscle. Co-expression of Rheb with p70s6k caused significantly increased 72

83 A GFP GST-p70S6K HA-Rheb eif2bε GST-tagged Endogenous p(t389)-p70s6k GST-tagged Endogenous p70s6k GFP Rheb HA-tag α-tubulin B. eif2bε (A.U.) GFP GST-p70S6K HA-Rheb Figure 6. p70s6k signaling does not alter total eif2bε cellular abundance in vivo. (A) Mouse TA muscles were transfected with GFP, GST-tagged p70s6k, and/or HA-tagged Rheb. Mice recovered for 48 h at which time muscles were harvested and analyzed by Western blotting with the indicated antibodies. (B) Values are means ± SEM for the eif2bε data shown in A, n=3-4 muscles / group. 73