Education, Faculty of
Kinesiology, School of
DSpace
UBCV
Li , Ching Wah Donna
Master of Science - MSc
University of British Columbia
A maximal eccentric (ECC) bout can confer protection by reducing exercise-induced muscle damage (EIMD) following a subsequent ECC bout, this is known as the repeated bout effect (RBE), a phenomenon that results in large non-specific translational and transcriptional responses. Resistance exercise training (RET) stimulates similar adaptations which becomes more refined with continuing training. It was hypothesized that RET and RBE may share a common mechanism involving remodeling of the extracellular matrix (ECM). Healthy young adults (6 female, 10 males; 20.9 4.1 years) performed 6 sessions of unilateral RET with the contralateral leg acting as a control prior to 2 bilateral bouts (B1, B2) of 300 eccentric knee extensions separated by 21 d. Bilateral muscle biopsies were obtained before and 48-hours after each bout. Muscle damage assessed as the area under the curve for isometric force loss over 72-hours following ECC, was attenuated in B1 by RET (42%±4.76, p=0.035). Protection conferred by B1, resulted in complete recovery of force loss by 48h. ECC elevated expression of Transforming Growth Factor-Beta (TGF𝛽, p=0.018) and Collagen Type IV (COL4A1, p=0.027) only in the RET leg after each bout, while Matrix Metalloproteinase-2 (MMP2) mRNA increased after ECC only in the control leg (p=0.049). These results collectively suggest that short-term RET modifies the transcriptional response of ECM regulators to bouts of high volume ECC exercise which may partially underpin the observed attenuation of ECC induced EIMD by prior RET. Further analysis is required to determine if altered transcription of ECM regulators is also reflected at the protein level.
SHORT-TERM RESISTANCE EXERCISE TRAINING ATTENUATES MUSCLE DAMAGE AND ALTERS TRANSCRIPTION OF MUSCLE EXTRACELLULAR MATRIX REGULATORS IN RESPONSE TO 300 ECCENTRIC CONTRACTIONS IN YOUNG ADULTS by CHING WAH DONNA LI B.A.S., University of Guelph, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Kinesiology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2022 © Ching Wah Donna Li, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Short-term resistance exercise training attenuates muscle damage and alters transcription of muscle extracellular matrix regulators in response to 300 eccentric contraction in young adults submitted by Ching Wah Donna Li in partial fulfilment of the requirements for the degree of Master of Science in Kinesiology Examining Committee: Dr. Cameron Mitchell, Professor, Kinesiology, UBC Supervisor Dr., Don McKenzie, Professor, Kinesiology, UBC Supervisory Committee Member Dr., James Markworth, Professor, Animal Sciences, Purdue University Supervisory Committee Member iii Abstract A maximal eccentric (ECC) bout can confer protection by reducing exercise-induced muscle damage (EIMD) following a subsequent ECC bout, this is known as the repeated bout effect (RBE), a phenomenon that results in large non-specific translational and transcriptional responses. Resistance exercise training (RET) stimulates similar adaptations which becomes more refined with continuing training. It was hypothesized that RET and RBE may share a common mechanism involving remodeling of the extracellular matrix (ECM). Healthy young adults (6 female, 10 males; 20.9 4.1 years) performed 6 sessions of unilateral RET with the contralateral leg acting as a control prior to 2 bilateral bouts (B1, B2) of 300 eccentric knee extensions separated by 21 d. Bilateral muscle biopsies were obtained before and 48-hours after each bout. Muscle damage assessed as the area under the curve for isometric force loss over 72-hours following ECC, was attenuated in B1 by RET (42%±4.76, p=0.035). Protection conferred by B1, resulted in complete recovery of force loss by 48h. ECC elevated expression of Transforming Growth Factor-Beta (TGF, p=0.018) and Collagen Type IV (COL4A1, p=0.027) only in the RET leg after each bout, while Matrix Metalloproteinase-2 (MMP2) mRNA increased after ECC only in the control leg (p=0.049). These results collectively suggest that short-term RET modifies the transcriptional response of ECM regulators to bouts of high volume ECC exercise which may partially underpin the observed attenuation of ECC induced EIMD by prior RET. Further analysis is required to determine if altered transcription of ECM regulators is also reflected at the protein level. iv Lay Summary Skeletal muscle is a dynamic tissue that responds to resistance exercise training (RET) to induce beneficial health effects (1). Following initiation of RET, adaptation occur in the skeletal muscle and the collagen network surrounding it known as the extracellular matrix (ECM) (2). An unfamiliar exhaustive exercise bout, particularly lengthening contractions, often results in such extensive change that protection is conferred to subsequent bouts (3) This is known as the repeated bout effect (RBE), a unique paradigm that can be used to understand short-term adaptations to resistance exercise (RE) (4). One mechanism thought to mediate RBE is ECM remodeling, which occurs in response to both RET and ECC contractions. Short-term RET likely stimulates ECM remodeling similarly to a single ECC bout. Therefore, it is of interest to determine whether RET can induce RBE, a phenomenon representing effective early adaptation and whether ECM-related molecular changes are similar between the two stimuli. v Preface The following thesis project was designed and developed by Donna Li with the assistance of Dr. Cameron Mitchell and the other students in the Aging, Nutrition, Exercise and Muscle Metabolism Lab. Scheduling, participant recruitment, and participant testing was conducted by Donna Li. Participant training was conducted by Donna Li and Rory Trevorrow. Analysis of results was conducted by Donna Li, Rory Trevorrow, and Ally Schweitzer with assistance from Dr. Cameron Mitchell. All aspects of the current thesis project were approved by The University of British Columbia’s Research Ethics Board (H21-01924). vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiii Chapter 1: Introduction ................................................................................................................1 1.1 The Skeletal Muscle and Its Extracellular Matrix .......................................................... 1 1.1.1 Adaptations ............................................................................................................. 2 1.2 The Repeated Bout Effect ............................................................................................... 3 1.2.1 Exercise-Induced Muscle Damage ......................................................................... 4 1.2.2 Characteristics of RBE ............................................................................................ 5 1.2.3 ECM Remodeling ................................................................................................... 7 1.2.4 Eccentric Model Limitations................................................................................... 8 1.3 Mechanisms of RBE ....................................................................................................... 9 1.3.1 Nervous System ...................................................................................................... 9 1.3.2 Myofiber and Tendon ........................................................................................... 11 1.3.3 Extracellular Matrix .............................................................................................. 13 1.4 Contribution of the ECM to Exercise-induced Adaptations ......................................... 14 1.4.1 ECM Stiffness ....................................................................................................... 14 vii 1.4.2 Collagen ................................................................................................................ 15 1.4.3 Transforming Growth Factor Beta ........................................................................ 16 1.4.4 Matrix Metalloproteinase ...................................................................................... 18 1.4.5 Transitional Matrix ............................................................................................... 21 1.4.5.1 Fibronectin ........................................................................................................ 22 1.4.5.2 Tenascin C ........................................................................................................ 23 1.5 RBE Adaptations .......................................................................................................... 26 1.5.1 Time Course .......................................................................................................... 26 1.6 Cross-Education ............................................................................................................ 28 1.7 Summary ....................................................................................................................... 30 1.8 Purpose .......................................................................................................................... 30 1.9 Hypothesis..................................................................................................................... 31 Chapter 2: METHODS................................................................................................................32 2.1 Participants .................................................................................................................... 32 2.2 Randomization .............................................................................................................. 33 2.3 Study Overview ............................................................................................................ 33 2.4 Initial Contact................................................................................................................ 35 2.5 Screening/Familiarization ............................................................................................. 35 2.6 Muscle Function Assessment ........................................................................................ 36 2.7 Resistance Exercise Training ........................................................................................ 36 2.8 Muscle Biopsy .............................................................................................................. 37 2.9 Eccentric Bout ............................................................................................................... 38 2.10 Sample Analysis............................................................................................................ 38 viii 2.10.1 Area Under the Curve for Isometric Force Loss ................................................... 38 2.10.2 TGFβ Protein ........................................................................................................ 39 2.10.3 RT-qPCR............................................................................................................... 40 2.11 Statistical Analysis ........................................................................................................ 41 Chapter 3: Results........................................................................................................................42 3.1 Exercise Performance and Muscle Function................................................................. 42 3.2 ECM-related Gene expression ...................................................................................... 44 3.3 Transforming Growth Factor Beta ................................................................................ 47 Chapter 4: Discussion & Conclusion..........................................................................................49 4.1 Main Findings ............................................................................................................... 49 4.1.1 Maximal ECC Contractions and RET as a Stimulus for the Repeated Bout Effect…… ............................................................................................................................. 49 4.1.2 ECM Remodeling in early adaptation to RET ...................................................... 54 4.1.2.1 Delayed Increase of Profibrotic Genes Following Maximal Eccentric Contractions ...................................................................................................................... 55 4.1.2.2 Transitional Matrix ........................................................................................... 58 4.1.2.3 Degradation of ECM by Matrix Metalloproteinase .......................................... 59 4.1.3 Prolonged response of the ECM to RET............................................................... 60 4.2 Limitations .................................................................................................................... 62 4.3 Future directions ........................................................................................................... 64 4.4 Conclusion .................................................................................................................... 65 Bibliography .................................................................................................................................66 Appendices ....................................................................................................................................85 ix Appendix A - Sample Muscle Function Testing Data Recording Sheet .................................. 85 Appendix B - Familiarization Session Data Recording Sheet .................................................. 86 Appendix C - Sample Muscle Function Testing Data Recording Sheet ................................... 87 Appendix D - Sample Training Session Data Recording Sheets .............................................. 88 Appendix E - Sample Food Log ............................................................................................... 89 Appendix F - Participant Information and Informed Consent Sheet ........................................ 90 x List of Tables Table 1. Participant baseline characteristics ................................................................................. 33 Table 2. Primer Sequences ............................................................................................................ 40 Table 3. Summary of Gene 3-Way ANOVA Interactions. ........................................................... 48 xi List of Figures Figure 1. Diagram of skeletal muscle and extracellular matrix ...................................................... 2 Figure 2. A theoretical model of the accumulation of adaptation induced by maximal ECC bouts and RET. ......................................................................................................................................... 7 Figure 3. Schematic of interactions within the ECM. ................................................................... 25 Figure 4. Indirect measures of muscle damage following 2 bouts of 300 ECC contractions ....... 59 Figure 5. Protection conferred by RET and B1 ............................................................................ 60 Figure 6. mRNA expression of COL1A1, COL3A1, and COL4A1 ............................................. 45 Figure 7. mRNA expression of FN and TN. ................................................................................. 46 Figure 8. mRNA expression of MMP2 ......................................................................................... 46 Figure 9. mRNA expression and protein levels of TGFβ ............................................................. 47 Figure 10. Schematic of conferred protection by RET and maximal ECC contractions. ............. 54 xii List of Abbreviations B1 Bout 1 B2 Bout 2 CON Concentric DOMS Delayed Onset Muscle Soreness ECC Eccentric EIMD Exercise-Induced Muscle Damage FN Fibronectin ISO Isometric MuSC Muscle Satellite Cells MTJ Myotendinous Junction MVC Maximal Voluntary Contraction MyoPS Myofibrillar Protein Synthesis RBE Repeated Bout Effect RE Resistance Exercise RET Resistance Exercise Training TN Tenascin C TGF Transforming Growth Factor Beta TGF-RII Transforming Growth Factor Beta Receptor Type 2 VL Vastus Lateralis xiii Acknowledgements Cam - Thank you for always providing me guidance, answering my often mindless questions, and always being in your office for impromptu meetings. I greatly appreciate the opportunity you’ve provided me and I am excited to see the exciting future that holds for the lab. Doctors - Dr. Khoele, Dr. Drapala, Dr. McKenzie, Dr. Prasad, Dr. Liu and Dr. Rau. Thank you for taking time out of your busy schedules and always having the best stories to tell! Participants - Thank you for enduring all 8 biopsies and over 4 hours of eccentric contractions on the Biodex. The Mitchell Army - Rory, Ally, Jordan, and Matt, thank you for always making yourselves available, all the things you’ve taught me, and all the help you’ve provided throughout the duration of the project. Justin and Dr. Whittaker - Thank you for letting me have access to the Biodex. Importantly, Justin, for pulling 13 hour days for no other reason than to let me use the Biodex. Can’t thank you enough! 1 Chapter 1: Introduction 1.1 The Skeletal Muscle and Its Extracellular Matrix In humans, skeletal muscles account for 40% and 30% of total body mass in men and women, respectively. Muscle fibers (also called myofibers) are multinucleated contractile muscle cells (or myocytes) which develop from myoblasts through a process known as myogenesis (5,6). Another cell type found in close association and vital for maintenance, repair and remodelling of the muscle fibres are satellite cells (MuSC). MuSC are quiescent stem cells that migrate from between the sarcolemma and basal lamina once activated to proliferate and/or differentiate to myoblasts, fusing with existing fibres (7). The basal lamina is the innermost structure of the extracellular matrix (ECM) and sheaths individual muscle fibers. Extending outwards are the three connective tissue layers epi-, peri-, and endomysium. The epimysium is primarily composed of Collagens Type 1, the perimysium Collagen Type I, III, V and VI, the endomysium Collagen IV, along with other matrix proteins (8–10). The endomysium wraps around each individual muscle fibre, the perimysium surrounds the bundles of myofibers, and the epimysium around the whole muscle (5) (Figure 1). Fibroblasts and/or myofibroblasts are the main cellular source of these collagenous components along with fibroadipogenic progenitor cells, stems cells that are a source of collagen and fibroblasts, its progeny (11). 2 1.1.1 Adaptations Adult skeletal muscle is relatively stable in normal conditions until disturbed from homeostasis by a stimulus such as exercise. Resistance exercise training (RET) leads to adaptations classically seen as increased muscle strength, cross-sectional area (hypertrophy), and alterations to the surrounding connective tissue (4,12,13). In the early weeks of RET, naive muscle experiences an initial rapid rise in strength, followed by increases in muscle size (14). It is thought that early strength changes are driven by neuromuscular adaptations followed by structural (12,15). A growing body of evidence indicates the significance of the skeletal muscle ECM in contributing to adaptations to exercise (10). This 3-D collagen scaffold plays an important role in Figure 1. Diagram of skeletal muscle and extracellular matrix 3 lateral force transmission, maintenance and repair of the muscle fibre, muscle structure, as well as regulation of myogenesis (10). Moreover, the ECM molecules respond to RET and appears to take part in exercise-induced adaptations, for instance, hypertrophy by influencing MuSC behaviour. Several groups have reported changes in ECM outcomes following less than 9 weeks of training in young participants (16–21). Increases are seen in mRNA expression of collagens (i.e., COL1A1, COL3A1), proteoglycans (i.e., ASPN, BGN), glycoproteins (i.e., TN, FN), as well as protein expression of collagens (i.e., Collagen Type I Alpha 1 & 2 Chain), glycoproteins (i.e., agrin, tenascin C) (16–21). Similar ECM transcriptional altercations were seen across training modalities including combined training, RET, and high-intensity interval training (HIIT). Upregulation of ECM-related molecules concomitant with classically seen adaptations is critical for muscle structure and stability. 1.2 The Repeated Bout Effect The repeated bout effect (RBE) is seen following a single eccentric (ECC) bout, whereby diminished exercise-induced muscle damage (EIMD), often quantified as strength loss, is observed. Higher peak force and lower motor unit recruitment during maximal and submaximal ECC contractions induce greater damage to contractile proteins, ECM, and sarcomeres. Greater mechanical stress is also imposed on each muscle fiber and crossbridge (22). Disturbed homeostasis from ECC contractions likely necessitates the activation of tissue remodeling (12). Whole-body molecular and cellular remodeling is not unique to RBE, but rather fundamentally evoked by all physiological perturbations. For that reason, RBE can be viewed as a constituent of skeletal muscle plasticity, an innate response to EIMD present to benefit functionality, a notion arising from Han Seyle’s’s general adaptation syndrome (GAS) (23). GAS 4 conceptualizes the innate tendency for adaptation in response to stress so that subsequent exposure to the causative agent does not exhibit alarm. Applied to exercise-induced adaptations, the resultant homeostatic alterations by EIMD likely prompt protection so that the same agent can be mitigated (24), hence lessened EIMD in subsequent similar bouts. 1.2.1 Exercise-Induced Muscle Damage EIMD is often associated with RBE and the two often come hand in hand. This EIMD sensation of weakness, discomfort or pain has been associated with connective and contractile tissue disturbance, and an increase in permeability of the sarcolemma (25). Stretching of the contracting muscle, specifically during the ECC component of a muscle action, develops substantial EIMD in the subsequent days after the cessation of activity (26). The current consensus suggests the magnitude of EIMD could be attenuated by exposing the skeletal muscle to an initial bout of the same (or similar) exercise protocol, with predominantly ECC contractions, if performed within 24 weeks of the first exercise bout (4,27,28). EIMD is indirectly indicated by strength loss, pain, delayed onset muscle soreness (DOMS), muscle edema, and restricted range of motion (27). Direct markers of EIMD are reflected by ultrastructural damage (i.e., sarcomere and myofibrillar disruptions) to the muscle observed by electron microscopy (29). The current gold-standard indirect marker for assessing damage is reduced muscle force (30). DOMS is a poor reflector of EIMD due to generally poor correlations with other indicators (31). EIMD can persist up to 14 d after the initial exercise bout, hindering both muscular function, movement, and athletic training, in turn impairing quality of life (32,33). In general, strength decrements are most prominent immediately after ECC, and restoration of force occurs 5 over the following ~7-14 d (34). Reductions in maximal voluntary contraction (MVC) of the muscle immediately postexercise are attributed to central and peripheral fatigue which recovers with rest (i.e., clearance of metabolites). At 24 h and onward, force decrements are mostly due to peripheral impairments including ultrastructural damage (35). Immediately and 1h after ECC contractions, isometric torque decreased to 40% of baseline values along with decreased voluntary activation indicating central fatigue. Changes in voluntary activation were not found 1 d postexercise, attributing continued MVC torque loss to peripheral factors (36). By 72 h, strength loss is likely only due to peripheral damage of the muscle fibers or by-products inducing weakness. The degree of damage that warrants regeneration and the time course of recovery are dependent on many variables, including the degree of strength impediment. Decreases of ~50% immediately after exercise typically require a minimum of 7 d until strength is restored back to normal (37,38). 2 weeks appear to be the average before baseline MVC is recovered (37). 1.2.2 Characteristics of RBE Protection towards a subsequent bout is not isolated to ECC contractions but can also be endowed by isometric (ISO) electrical stimulation, non-damaging ECC, and ISO contractions at long muscle lengths (3,39–41). Currently, little work within the RBE paradigm has addressed RET as a damaging stimulus or directly compared it against acute ECC protocols. RE generally does not produce the same level of damage, however, repeated RE bouts can also attenuate EIMD (12). Conventional RET can also induce EIMD in the naive muscle (12,35,42). High-load RE (~70% 1 RM) has been shown to decrease muscle strength to a similar magnitude as 300 maximal ECC, ~20% decrement at 24 h in healthy untrained young adults (4,12). Though, 6 previous studies have exemplified that EIMD is not a prerequisite for RBE (43). Rather, the magnitude of protection conferred is dependent on volume, intensity, velocity and muscle group length (28). Intensity and training load do not independently predict protection. While 40% ECC in the elbow flexor conferred half as much protection as 100% ECC, the intensity-dependent nature does not hold true if the submaximal exercise is repeated (44). Four bouts of 40% ECC were sufficient for mimicking an initial 100% ECC bout (45). Thus, the potency of protection cannot be inferred from one variable alone. Non-damaging submaximal exercises preceding maximal ECC by at least 2 d can provide a preconditioning effect by decreasing isometric force loss and accelerating recovery (39,43). Attenuation of damage by 20% ECC and 20 degree ISO contractions of the elbow flexor performed prior to a damaging bout persisted up to 4 d post max ECC (46). Even two ISO contractions performed 2 d prior to 30 ECC contractions conferred protective effects to the elbow flexors (39). In the lower limbs, low-intensity ECC contractions of the knee flexor performed on standard exercise machines (3 sets of 10) attenuated the dependent variables, muscle soreness, peak CON-torque loss, range of motion (ROM), creatine kinase (CK) activity and myoglobin concentrations, when 30 maximal ECC bout was performed 2 d (50% protection) or 7 d later, with the effect decreasing between these two time points. The protective effect was abolished 14 d postexercise (40). The low-intensity ECC did not cause any changes in the dependent variables (e.g., MVC, ROM, CK activity, myoglobin concentrations) but still protected from maximal ECC contractions, albeit only 30. Nevertheless, it can be concluded from these results that even when the initial bout was non-damaging, validated by no significant increase in damage markers, protection was still conferred between 7-14 d. Most studies only perform one bout of the preconditioning exercise. Repetition of the same preconditioning 7 exercise, such as in RET, may summate to increase the duration or magnitude of protection, considering maximal ECC lends attenuation up to 24 weeks (27) (Figure 2). Moreover, prolonged and repeated non-damaging RE is sufficient in alleviating EIMD by the second week of bilateral leg press and extensions performed twice weekly (12). This attenuation is likely representative of structural and functional adaptation similar to that of RBE, with the difference being a cumulation of intermittent protection conferred by each training session to simulate one ECC bout. Moreover, alleviation of damage is followed by quantifiable adaptations, classically seen as increased cross-sectional area and increased strength (47). 1.2.3 ECM Remodeling Recently, ECM remodeling has been identified as a mechanism of RBE (4,48). RBE is likely a derivative of long-term adaptation, whereby remodelling induces protection from EIMD. Distinct ECM reconstructive activity, increases in collagen and growth factors, frame exposure Figure 2. A theoretical model of the accumulation of adaptation induced by maximal ECC bouts and RET. 8 to the second damaging bout, potentiating its contribution to protection (4). While molecular signatures of skeletal muscle adaptation to RET have been well studied, literature regarding the ECM is lacking. Unveiling molecular guides of the early phases in adaptation can further understanding of when acute changes translate to long-term adaptations, to elucidate “why”, “how” and “when” the skeletal muscle and its ECM submits itself to reassembly, and identify the role ECM plays to attain the abundant beneficial effects associated with exercise. 1.2.4 Eccentric Model Limitations An ECCcontraction produces higher forces at a lower energy cost compared to shortening actions, CON contractions, but at the cost of greater disruptions to the myofiber (49,50). The generally accepted dogma, that acute maximal ECC contraction leads to EIMD, has steered the direction of research on repair and remodeling to utilize this premise to investigate adaptive mechanisms in response to novel exercise in humans. ECC contractions on a dynamometer are often used to produce RBE and damage, but different types of exercises can be used including downhill walking/running/stepping, submaximal ISO or ECC contractions as a percentage of maximal (100%) ECC, or electric stimulation (28). Use of damaging bouts in the investigation of exercise response raises several caveats. 1. It is not reflective of most actions performed during exercise situations 2. Unable to discriminate RBE as an intrinsic property of ECC muscle action or the result of unaccustomed exercise 3. Degree of damage in laboratory testing is difficult to replicate even during intense exercise bouts. Most advances in the understanding of RBE mechanisms primarily use the elbow flexors as the muscle of analysis with little data on the lower limbs (51). Since elbow flexors and extensors demonstrate greater susceptibility to EIMD, findings cannot be directly extrapolated to the lower limbs. Applying RET in the RBE paradigm 9 may resolve several of these caveats considering its conventional applicability, incorporation of both CON, ISO and ECC in movements, and replicability of damage in real life. RBE can be induced by a number of preconditioning exercises, however, it is not known whether repeated RE bouts can replicate an acute ECC bout. RET is a commonly performed mode of exercise and can attenuate EIMD when repeated. Mechanistically, the two likely follows similar kinetics, thus demonstrating RBE using RET, especially in the lower limbs, will enhance knowledge surrounding both mechanisms of early adaptations to exercise and why protection is conferred when EIMD occurs. 1.3 Mechanisms of RBE Despite the ubiquity of RBE in observation, the mechanism of this protective process is not well understood. Three potential sites of adaptations have been proposed, the nervous system, myofiber, and the ECM. It is unlikely that one mechanism can independently explain all RBE observations, but whether the mechanisms amalgamate or remain mutually exclusive will require research to delineate. Importantly, of the pathways outlined below, the paucity of ECM driven mechanisms and adaptations in response to exercise requires further exploration to supplement the current mechanistic understanding of this protective effect. 1.3.1 Nervous System The first proposed and earliest attributed mechanism is the neural theory. During the early phases of training, a disproportionate increase in muscular strength to mass is observed (52), this is attributed to training-related neural adaptation at the level of the motor cortex, spinal, and neuromuscular junction (53). Similar strategies are thought to be employed in RBE after one bout including alterations in motor unit recruitment towards low-threshold motor units, less 10 damage to fast-twitch fibres and/or overall fewer units, less damaged fibers, to achieve the same production of force in the subsequent bout (51,54). Synchronicity of firing to coordinate synergistic muscles, consequently distributing workload over a greater motor unit pool while maintaining force production (55). The proportion of neural contribution to RBE has been questioned by several studies reporting the presence of RBE in absence of change in electromyography (EMG) parameters (56). McHugh and colleagues did not observe a difference in EMG activity between the initial and subsequent bouts of 60 ECC knee flexion despite evident RBE (27). Although this may be due to the low number of ECC performed. Ye and colleagues found that trained men experienced similar strength loss of the biceps brachi immediately following maximal CON and ECC exercises whereas their untrained counterparts experienced greater loss to ECC exercise and less to CON (57). Reduction of EMG parameters was similar in both groups suggesting this advantage was not linked to training-related neural adaptations. These findings are contradicted by neuromuscular changes in physically active young men and women, following 6 weeks of CON or ECC training (58). Both groups demonstrated increases in peak torque values despite similar force decreases immediately postexercise. Long-term neural adaptations may play a greater role in strength developments, but not to alleviation of fatigue immediately postexercise. However, EMG analysis presents issues such as the difficulty of removing noise contamination, reduction of EMG quality during filtering, etc. Recent advancements were not applied in these studies and results cannot fully dismiss neural modifications as a mechanism. 11 1.3.2 Myofiber and Tendon Myofibril adaptations largely involve architectural remodeling and a modified acute inflammatory response with decreased infiltration of macrophages (59). Overstretch of uneven sarcomeres in the naive muscle is thought to be the cause of EIMD (26,60). Architectural remodeling by ECC contraction involves the increase of fascicle length by addition of sarcomeres in series, causing a shift in the optimal angle and its range for production of maximal force which reduces mechanical strain at long muscle lengths (61,62). However, changes in optimal angle do not last for more than 4 weeks while RBE can last beyond 4 weeks (44). In further support, changes in fascicle length of the medial gastrocnemius were not observed between the initial and subsequent bout of backward downhill walking separated by 7 d suggesting sarcomerogenesis is unlikely responsible (63). Tendon compliance is another potential mechanism and greater strain is posited to have greater effects on tendon adaptations. However, results of several studies have been conflicting. Some studies found no change in Achilles tendon stiffness following eccentric heel drop training (64,65), while others found a decrease after 7 weeks of RET (66). Mechanical properties of other muscle groups may differ and muscle and tendon behaviour may be altered differentially. More research is required to determined how stiffness is altered by repeated eccentric bouts. Mechanical stress can damage the excitation-contraction coupling machinery, inaugurating a loss of calcium homeostasis that initiate a phagocytic phase (67,68). Subsequent strengthening of the cell membrane, whether by increased tolerance of calcium fluctuations or permeability may protect from calcium homeostasis disturbances and the ensuing proteolytic response (54,69). Calcium plays a multifaceted, pivotal role in the skeletal muscle, muscle contraction and ATP synthesis in the skeletal mitochondria. Acute mitochondrial calcium 12 overload in response to ECC contractions has been shown to lead to mitochondrial dysfunction, but can be ameliorated by training (70). Reduced mitochondrial calcium content 48 h post-acute ECC exercise observed in rats who received exercise-specific training prior to the first ECC bout reduced mitochondrial protein cell death, improving mitochondrial respiratory function (71). Sarcoplasmic reticulum calcium handling has also been shown to improve with training. Gekl and authors reported an increase in sarcoplasmic reticulum calcium release and no change in uptake when athletes perform repeated high-intensity exercise training programs (72). Enhanced calcium handling by the sarcoplasmic reticulum, improved release rates which prevent muscle fatigue and aid contractility. This enhancement may be pertinent to preventing disturbances in calcium homeostasis. Calcium disturbance may also impact muscle stiffness. Application of one maximal ECC contraction bout appears to elevate stiffness, shear elastic modulus (µ) the index for stiffness, up to 21 d in various regions of the elbow flexors (73). In the lower limbs, stiffness of both the rectus femoris and VL increased significantly at 24 h post loaded downhill walking and rectus femoris µ remained elevated for 2 weeks (74). The authors proposed elevated µ was a result of disturbed calcium homeostasis and/or edema (74). The degree of increase in µ is associated with degree of force loss, and a second ECC bout attenuates degree and duration of increased µ (73,74).Taken together, the excitation-contraction coupling machinery can adapt to exercise stimulus by maintenance of calcium regulation to protect against proteolysis. But, excitation-contraction impairments are most prominent immediately after exercise making its involvement in diminished force loss days following an ECC exercise minute. Although long-term training may deter excessive EIMD by higher control of calcium movement (75). Heat shock proteins (HSPs) are vital in the stress response for the protection of protein denaturation for cell survival and may also be involved to protect against aggravated stress (76). 13 Acute increases have been observed post ECC or running exercises (77,78), but after a repeated bout 8 weeks later, HSP content was attenuated in a dose-response manner dependent on the level of EIMD, indicating a diminished stress response (79). Modification of the acute inflammatory response to a more potent, targeted inflammatory response could explain the quicker recovery of damage markers in subsequent ECC bouts (80). 1.3.3 Extracellular Matrix ECM adaptations as an explanation for RBE stems from the notion that EIMD occurs when ECM disruption fails to support muscle integrity, and increases in intramuscular connective tissue or change in dynamic and passive muscle stiffness may augment the damage response (48). The importance of this 3-dimensional network binding the skeletal muscle has been previously highlighted, though the role it plays in RBE has yet to be defined. In recent years several studies have identified the dynamic reciprocity between the muscle cells and the microenvironment in the modulation of tissue repair, maintenance and function (81). The ECM is instrumental for the integration of biochemical and mechanical signaling, acting as a local repository for matrix components. It can bind and localize signaling molecules (i.e. growth factors – TGFβ, CTGF) for presentation to cells of the niche (82). Cross-talk, the autocrine/paracrine action of local cytokines and GFs, and cellular sensing of their surrounding elasticity may be primary drivers of exercise-induced adaptations (83). ECM remodeling appears to be biphasic, first with an upregulation of glycoproteins, termed transitional matrix, followed by structural components such as collagen (4,84,85). Concerted synthesis and degradation of the ECM is key to exercise stimulated remodeling. Exercise can increase both collagen production as well as MMP activity (86). Turnover is 14 mediated by pro-fibrotic factors (e.g., TGFβ, CTFG), MMPs, and a supportive regenerative environment. Control of these components and the time course of ECM remodeling has yet to be detailed in the literature. 1.4 Contribution of the ECM to Exercise-induced Adaptations Research has identified ECM remodeling as one potential mediator in the adaptation of the skeletal muscle and its microenvironment to mechanical stimuli (85). Besides maintaining muscle integrity and structure, the ECM also adapts to muscle growth, directs repair processes, and transmits lateral contractile forces to the tendon and other muscle fibres (87,88). 1.4.1 ECM Stiffness Approximately ~50% of longitudinal stress generated during muscle contraction is transmitted to the fascia, along with longitudinal strain, and radial stress and strain (89). Stress is the internal forces acting on the muscle independent of external load, and strain is the deformation of the muscle. Longitudinal stress or myofascial force transmission allows for the transmission of force through the muscle fiber to the surrounding ECM (90). When the muscle shortens, the increase in diameter radially pushes the ECM outwards, creating radial stress. Prolonged, repeated activation and relaxation of the muscle may induce ECM remodeling to accommodate applied stress (89). Hypertrophy of the muscle with chronic RET may also induce similar stress. There have been mixed reviews in literature of whether the ECM becomes stiffer or increasingly flexible after exercise or training as it is difficult to separate the muscle and the connective tissue in vivo (34,89). Stiffness of the whole myofiber isolated from animal biopsies can be quantified by a technique called atomic force microscopy indentation used for extraction of the Young’s Modulus which is the ratio of stress to strain (91). One study that have 15 successfully isolated the ECM measured stiffness by calculating the ratio between length after stretch to initial length (92). It has been previously reported that passive stiffness increases with ECM remodeling so that the muscle is protected from mechanical stress. However, evident RBE without such mechanical changes suggests this increase does not dictate adaptation (63). Demands of different movement may determine ECM elasticity after remodeling, but it is not known which state is “better” given its complexity. Defining the ECM by “stiff” and “soft” oversimplifies the number of factors that play into this multifaceted structure. Nevertheless, ECM elasticity is paramount for cell mechanosensing of their surrounding environment, demonstrated to have a critical effect on cell fate especially in the scope of regeneration (93). Substrate stiffness in vitro can mediate, for instance, MuSc, promoting apoptosis of migratory cells on softer substrates (94). Elevated µ after exercise may be a factor of TGFβ by enhancement of fibrotic factors, thereby increasing MuSC migration and differentiation. Stiffness of the ECM may modulate cellular events and development of methods to detect substrate stiffness in vivo will elucidate potential exercise induce changes and how they affect cell fate (95). 1.4.2 Collagen The endomysium is primarily composed of Type I and III collagens, while collagens type IV and VI make up the basement membrane (96). Force generated by cross-bridge formation is transmitted from the fibre to the basement membrane, then collagen I and III, and finally the tendon (97). Collagen gene expression transiently increases as early as 1 h postexercise (98,99). Specifically, animal models of downhill running depict an increase of COL1A1 and COL3A1 1 d, and COL4A1 6-12 h after exercise, with peak levels observed at 2-4 d (100,101). Interestingly, 16 COL4A1 appear to negatively correlate with the severity of damage. The most damaged part of the quadriceps femoris muscle decreased concentration 1 and 2 d post running, returning to control level by day 7 when damage has dissipated (101). With training in rats, ECC and ISO exercise increased COL1A1, while COL3A1 responded to ECC, isometric and CON training (102). 12 weeks of full body endurance and strength training in humans increased COL1A1, COL3A1, and COL4A1 by 140%. Of all the genes that were upregulated, 20% was ECM-related (20). A single bout of ECC contractions increased the staining intensity by Serius Red staining of type IV collagen to a moderate to large effect size at 22 d post exercise while no change was seen in types I and III collagen staining intensity. Relative total endomysial collagen did not change between preexercise and day 22 values (3). Collagen genes evidently responds to exercise, but does not directly translate to total collagen indicating either elevated turnover rates with increase breakdown or low post-transcriptional regulation. 1.4.3 Transforming Growth Factor Beta Stress-responsive factors can influence the turnover of ECM proteins (103). TGFβ is a multifunctional cytokine implicated in EIMD and regeneration for the preservation of ECM equilibrium, inhibition of myogenic responses, facilitation of the inflammatory response and stimulation of fibrosis (104,105). Fibroblast phenotype and biological activity are dictated by TGFβ to deposit matrix proteins and stimulate profibrotic genes (e.g., collagen and CTGF). This cytokine can also stimulate the production of matricellular proteins (e.g., FN), and regulate MMP-induced ECM degradation through its ability to stimulate synthesis of both MMPs and their inhibitors (106). While critical for the rebuilding of the ECM, overexpression can promote 17 fibrosis affecting muscle function negatively, but inhibition of TGFβ leads to impaired structural remodeling despite rapid recovery in strength (106,107). Produced by various cell types, including T cells, macrophages and fibroblasts, TGFβ is secreted into the ECM in its inactive form bound to a latency associated peptide (LAP), freely or attached to other matrix proteins such as fibronectin (FN) (108–110). Mechanical stress can induce ECM degradation and signal directly via LAP’s integrin interaction, to allow the release of sequestered TGFβ into the extracellular space (111). Integrin-mediated adhesions allow cells to directly sense the elasticity of the ECM and the tension-dependent signaling nature of TGFβ means matrix stiffness can modulate levels of active TGFβ (82). According to the model proposed by Hinz and colleagues, a stiff substrate can resist deformation under stress which can then induce a conformational change in LAP’s integrin handle resulting in increased TGFβ (82). Since muscle stiffness is elevated 2 weeks after a bout of downhill walking, it is likely that this is the period of increased active TGFβ (112). Bioavailability of TGFβ is regulated by proteolytic liberation from LAP by enzymes including MMP2 or 9 (113). Although TGFβ is often associated with collagen production and fibrosis, the exact mechanism is unknown. In cultures of rat cardiac fibroblasts, TGFβ did not directly stimulate collagen production, but rather the irreversible differentiation of fibroblasts to myofibroblasts, increasing collagen producing activity (114). The myofibroblastic phenotype is quite stable, likely due to high concentration of secreted TGFβ acting in an autocrine manner (115). These data agree with the central role of TGFβ in collagen deposition and promotion of a fibrotic phenotype (114). TGFβ is a chemoattractant for fibroblasts, instigating recruitment to site of injury and subsequent collagen production. Another proposed mechanism is TGFβ’s negative 18 regulation of microRNA-29, a noncoding RNA sequence capable of inhibiting translation of collagen-related target genes (116). These mechanisms indicate an association between TGFβ and collagen production. Gene expression of TGFβ has been shown to increase in both animal and human muscles after ECC exercise in relation to the inflammatory response (117,118). As a potent chemotactic factor, local production recruits neutrophils and monocytes to sites of EIMD to support the acute inflammatory response. Its production is further amplified by release from resident macrophages and infiltrated leukocytes (119). In the later phases of regeneration, TGFβ suppresses inflammation (120). Trained rats performing an acute bout of exercise demonstrate a transient decrease in TGFβ mRNA at 0 h (121). When compared to their untrained counterparts, preexercise values of TGFβ mRNA and protein increased and decreased to 6 weeks of training, respectively (121). The authors concluded that physical activity may be a potent stimulus for TGFβ at the gene level, but this increase does not translate to increases in protein. In humans, maximal ECC contractions appears to increase the receptor, TGFβR-II, at 72 h and 27 d postexercise, but not at 2 d (4,80). While decreased protein levels of active TGFβ is observed in animal models, no study has reported on exercise effects of the protein in humans (121). Upregulation of the TGFβ signaling pathway may be a function of increased ligand or demand for downstream products, such as collagens. 1.4.4 Matrix Metalloproteinase One major determinant in ECM-related adaptive modifications is MMPs. This family of zinc- and calcium-dependent enzymes are responsible for direct proteolytic degradation and indirect synthesis of ECM molecules (i.e., proteolytic activation of pro-fibrotic genes), such as 19 the liberation TGFβ from LAP. Different sub-families of MMPs are distinguished by function. 23 known MMPs have been identified in humans, three collagenases (MMP-1, -8, and -13), two gelatinases (MMP-2 and MMP-9), three stromelysin (MMP-3, -10 and -11), six membrane-type (MMP-14, -15, -16, -17, -24 and -25), two matrilysin (MMP-7 and -26), and other (MMP-12, -19, -20, -21, -23A/B, -27, and -28). MMPs’ biological activity is regulated at several levels. First, gene expression is controlled by numerous factors, including growth factors, cytokines, and interactions with ECM components (122). Thereafter, the enzyme is secreted as an inactive zymogen, pro-MMP. Cleavage of the propeptide domain by MMP2, MMP13, MMP3, MMP9 among other proteinases yields active MMP (123). The functionality of MMPs is not limited to ECM remodeling, they facilitate cell migration, alter the ECM micro-environment, modulate activity of biologically active molecules by direct cleavage of inhibitors, and liberation of molecules anchored to the ECM, and vascularization (113,123,124). MMP2 and 9 are the major MMPs for the breakdown of collagen IV and V, the main component of the basement membrane, as well as FN, proteoglycans and laminin (11,124). The primary inhibitory enzyme for MMP2 and MMP9 are TIMP2 and TIMP1, respectively. Their ability to degrade the basement membrane facilitates cell migration (125). For instance, MuSC that reside between the sarcolemma and the basement membrane is allowed across the basement membrane for subsequent differentiation and proliferation, a key step in muscle regeneration (126). MMP2 is ubiquitously expressed in human skeletal muscle and constitutively expressed on the surface of myofibers and fibroblasts in normal adult tissue (127). In vitro studies indicated that around ~30% of total MMP2 was associated with the ECM, the remainder inside the muscle fibers and freely diffusible (128). Formation of a MMP14 homodimer facilitates 20 activation of MMP2 to an intermediate form prior to autocatalysis (129,130). Otherwise, pro-MMP2 can be activated by thrombin and activated protein C (131). Evidence further suggests a potential link between MMP2 and long-term RET-induced ECM remodeling. Specifically, in MMP2 KO mice, impaired ECM remodeling is concordant with a reduction in hypertrophy and force production (132). MMP9 is stored in intracellular secretory granules within neutrophils and remains largely dormant in absence of stimuli (127). Only upon tissue injury do MMP 9 gene expression and activation increase to target collagen I, III, V, FN, and TGFβ degradation (133). The time course of expression is dictated by the stage of remodeling. In animals, MMP9 appears to be extensively upregulated in the immediate period following injury and drops off after 3 d. Whereas, MMP2 continues to be upregulated up to 10 d post-lesion, concurrent with formation of new fibers (134,135). The differential pattern of expression could be that MMP9 is stimulated by acutely responsive proinflammatory factors, whereas MMP2 is correlated with myofiber regeneration (134). Human studies demonstrate a similar pattern in both muscle and plasma. Young participants subjected to ergometer cycling had increased muscle protein levels of MMP9 immediately following and 120 min after exercise. MMP2 levels were not significantly different to preexercise values (136). In a later study by the same authors, unilateral dynamic constant-load knee extension with either blood flow restriction or without did not change muscle MMP2 levels until 10 d after training and levels remained elevated until cessation of training at 5 weeks (137). Conversely, MMP9 transcript only increased significantly after the first bout of exercise. Zymography analysis for protein levels after a single bout of exercise correlated with mRNA levels, no change was seen in MMP2 and MMP9 increased (136). This was further supported by immunohistoreactivity of MMP9 surrounding the muscle fibre. No MMP9 staining was evident in the muscle fiber, whereas MMP2 was found both surrounding and within the 21 muscle (137). In sum, these findings indicate that MMPs dynamically respond to both acute exercise and training. They may play a role in regulating inflammation, by MMP9, or subsequent regeneration by MMP2. Ultimately, balance between ECM synthesis, MMPs, and their inhibitors, tissue inhibitors of metalloproteinases-1/2 (TIMP-1/2), plays a central role in regulating overall homeostasis (138). The amount of connective tissue produced must be finely tuned as dysregulation leads to pathological conditions – fibrosis, matrix weakening and others (139). Thus, similar to muscle hypertrophy, the net collagen balance may be key for defining the role of exercise-induced ECM remodeling. 1.4.5 Transitional Matrix The composition of the ECM matrix changes with respect to the demands of the cellular niche. A local, transient upregulation of a glycoprotein transitional matrix has been observed upon injury (10). ECM remodeling seems to occur in two phases (4,84). In studies using the amphibian limb regeneration models, early regenerative stages depict a downregulation of ECM proteins, such as laminin and type I collagen, along with a striking upregulation of TN, FN, and MMPs (30,140,141). Subsequent upregulation of collagens completes the biphasic remodeling of ECM (23). The biphasic model likely follows the sequence of a proinflammatory response for clearance of debris and a later rebuilding phase. In humans, this regenerative matrix provides instructional cues in the embryonic environment. The temporally coordinated deposition of TN and FN could directly affect muscle cell behaviour and direct subsequent remodeling. 22 1.4.5.1 Fibronectin FN is a cell surface-associated glycoprotein normally found within the interstitial matrix of the epi-, peri- and endomysium (142). It is also often co-localized with TN at the myotendinous junction (MTJ) (143). During repair and/or regeneration FN is upregulated by various cell types including fibroblasts for cell adhesion and matrix assembly (142). Within muscle cells, cytoskeleton contraction occurring alongside muscle contractions can direct FN to assemble into fibrils (144–146). In vitro studies have demonstrated the elasticity of FN allows for cytoskeleton contraction at higher amplitudes (147). Computation models illustrating the interaction between actomyosin machinery and external signals exhibit a force limiting phenomenon of stiffer surfaces due to the inelasticity of the environment (148). Fibrillogenesis may contribute to decreased EIMD during higher amplitudes of muscle contraction due to increased elasticity. FN is important for supporting an adhesive environment. FN may also participate in physically directing collagen and MuSC as it is the preferred adhesion substrate through integrins embedded in the myofiber membrane (142,149,150). When mouse MuSC-derived and human myoblasts were seeded on arrays containing 36 different ECM conditions, there was a pronounced preference for spots containing FN (150). Furthermore, knockout-FN models of adult MuSC lead to reduced proliferation and symmetric divisions suggesting initial adhesion may be a critical step (151). During matrix assembly, FN can tether together cell surface receptors and a variety of compounds including collagen and growth factors, which may promote physical communication between cells (152). Appearance of FN in the transient transitional phase may be to guide cellular action and bring together molecules in an orderly manner. In 23 healing skeletal muscle wounds, the fibrin-fibronectin cross-link optimizes the adhesion of fibroblasts (153). Early phases of granulation are marked first by the appearance of FN, followed by collagen Type III (154). During the development of the granulation tissue, FN is thought to be important for the degradation of collagens by adherence to collagen digesting enzymes (155). While damage by exercise is not comparable to that of injury, FN may similarly promote collagen degradation by MMPs. Importantly, FN has been shown to respond to exercise. In response to 100 ECC contractions, FN immunoreactivity increased significantly from 5.6% to 12.8% of muscle cross-sectional area (84). 12 weeks of strength and endurance training in inactive middle age men increased FN by 1.4 fold (156). It seems that FN responds to both acute exercise and training. This increase could be to support a regenerative adhesive environment and increase the elasticity of the ECM. 1.4.5.2 Tenascin C Tenascin C (TN) under normal conditions is confined to the MTJ (157). TN is not produced by fibroblasts within the muscle, rather, it is only synthesized by fibroblasts located in proximity to the MTJ or by MuSC to promote MuSC expansion for muscle repair (157,158). The molecular mechanism by which it can do so remains unclear. Regulation of TN has been conflicting. Tensile stress has been postulated to activate TN at a transcriptional level in a dose-dependent manner (159,160), however, transient upregulation of newly synthesized TN following injury suggests multiple regulatory stimuli (161), though disruption of the ECM appears to be the point of intersection. Upon injury, inflammation or mechanical stimulation, this adhesive-modulating ECM molecule facilitates a de-adhesive environment for cell motility and 24 proliferation contrary to FN (4,103). A de-adhesive environment in the ECM, is thought to be an intermediate state favourable for cell motility and proliferation. In a genome-wide transcriptomic analysis after ECC contractions, several high responding genes corresponded to the ECM, one being TN which increased 11.6-fold (162). Rapid, upregulation of TN with more than a 10-fold change in gene expression is observed earliest, 5 h post voluntary ECC contractions (163), and remains elevated at 8 d (157). Levels return to baseline by 27 d (4). Actualized, TN immunoreactivity markedly increased 48 h after the first bout of 300 ECC contractions and was attenuated by the second ECC bout (4). Lower volume ECC contractions also appear to increase muscle immunoreactive for TN after 100 ECC contractions and low volume ECC exercise at 24 h, and 2 d and 8 d postexercise, respectively (84,164). Appearance of TN to low volume exercise, such as kicking at 67% of max workload, supports that injury to the muscle is not a necessary stimulus (163). Evidently, both FN and TN are prominent during EIMD recovery. However, their opposing functions question their temporal coordination. It seems that counterbalancing the properties of FN and TN mediates endothelial cell adhesion, however, further research is required to access the mechanistic relationship between these two matrix components in skeletal muscle (165). Additionally, whether such a transitional matrix is required in adaptations to RET is unknown. RET does not elicit the same level of damage as maximal ECC, but even then, migration of MuSC still occur for enhancement of muscle hypertrophy. A comparison of the two exercise modules will hopefully answer whether the invasion of matrix proteins is essential or only necessary when damage is present. 25 Interaction of these ECM components is illustrated in Figure 3. Within the ECM, MMP2 and 9 both degrade Collagen IV and V. MMP2 contributes to the liberation of latent TGFβ while MMP9 can breakdown active TGFβ. Active TGFβ can signal through TGFβR-II to increase collagen synthesis. In the matrix, FN and TN work in opposition to produce an adhesive and deadhesive environment, respectively. Figure 3. Schematic of interactions within the ECM. 26 1.5 RBE Adaptations Provided that the myofiber and ECM work intimately together through cross-talk, their repair and modulation kinetics should follow a similar course for exercise-induced adaptations. Since skeletal muscle is bound by the ECM, hypertrophy is probably accompanied by ECM remodeling to accommodate increasing myofiber size (74). Coalescing the works of Damas and colleagues on the protein synthetic response in relation to hypertrophy together with molecular ECM changes after damage by the Hydahl group (4,12), RBE adaptations to a single damaging bout likely resemble that of early adaptations to RET. 1.5.1 Time Course Discrepancy between collagen synthesis rates and transcriptional levels may arise from the discordance of acute and chronic demands of the muscle architecture. Acute changes in myoPS cannot predict chronic hypertrophy until cessation of EIMD (12). Continuing with the comparison to hypertrophy, long-term RET induced muscle mass gains are related to enhanced change in synthesis rates, translational efficiency and translational capacity, ribosome biogenesis augmented resting myoPS (166). A similar explanation may be used for the elevated collagen expression at 27 d post-ECC. TGFβ has been shown to promote fibroblast proliferation and myofibroblast transdifferentiation, in vivo and in vitro, respectively (167,168). Elevated levels of TGFβ may increase collagen synthesis by increase the number of fibroblast or myofibroblast numbers. Initial secretion of collagen is likely not transcriptionally regulated while delayed synthesis may be an outcome of expanded collagen producers. Effects of RBE diminish during 4 maximal high volume ECC bouts, and the protection conferred by each subsequent bout is smaller than that of the initial bout (45). This may indicate the refinement of adaptation. ECM remodeling differs from hypertrophy in the sense that it 27 cannot continuously increase in size. Alike skeletal muscle where breakdown and synthesis act as quality control, continued elevation of structural collagen and MMPs may be indicative of accelerated remodeling with RET (4).12 weeks of RET increased collagen mRNA expression in rats, but the overall percentage of connective tissue decreased, possibly due to greater turnover (169). Immediately after RE, high levels of the collagen degrading enzyme, matrix metalloproteinase-9 (MMP-9), correlates with the acute inflammation seen postexercise (170). ECM degradation by MMP-9 may be an antecedent event to the novel synthesis of ECM proteins. Effective remodeling appears to be key for a healthy ECM, as accumulated collagen content in aged muscles impairs functionality (150,171). Exercise is able to ameliorate age-associated fibrosis driven by weak degradative capacity of misfolded and accumulated ECM (172). Thus it seems that exercise increases overall turnover of ECM proteins, but further corroboration will be required to define long-term ECM adaptations to RET or continuation of ECC bouts. ECM characteristics have been shown to regulate MuSC function and stemness (10). Regenerative capacity can be enhanced by softer substrates, therefore, initial low levels of collagen or its rapid clearance may support MuSC migration across the basement membrane (173). The ‘cocktail’ of biochemical cues released during these initial phases, for instance, TN or FN of the transitional matrix, can direct MuSC adhesion, differentiation and proliferation (174). ECM remodeling is likely biphasic. Beginning first with repair then remodeling. Different ECM molecules contribute to these two phases. Synthesis and degradation maintains equilibrium in the ECM, while greater myofibrillar protein synthesis (myoPS) promotes hypertrophy(10). Different ECM molecules contribute to these two phases. Therefore, the absence of correspondence 28 between early and eventual modifications in both collagen and muscle proteins may be fundamental to exercise-induced changes. 1.6 Cross-Education Inter-subject differences - genetics, history and maturation - introduce variability to ECC-induced damage markers. Deviations in mean muscle response are especially evident in small sample sizes (175–177). A unilateral exercise model can reduce confounding factors, cost and duration, and increase statistical power (178). One common criticism of within-subject designs is the transfer of skeletal muscle adaptation from the trained limb to the contralateral (CL) untrained limb. This phenomenon, cross-education, has been well documented in exercise physiology (179). The existing literature on cross-education largely probes strength transfers. The typical magnitude revealed by one meta-analysis, was 7.6% on the contralateral (untrained) limb when 35.1% of strength was gained on the ipsilateral limb (trained) (180). Another meta-analysis reported a 12% gain from baseline, ranging from 8-18% (181). In addition to the widely observed CL strength enhancements, unilateral damaging bouts can also confer protection from EIMD to the CL limb (27). From a single maximal eccentric bout of the ipsilateral elbow flexor, CL-RBE can be observed up to 8 weeks with the protective effect decaying temporally (182). In the knee flexors, CL-RBE appeared to be more transient, dissipating before 28 d. However, CL-RBE is shown to be absent in response to some protocols such as an eccentric step-down protocol as evidenced by no protection in strength or tenderness in the CL leg (27). This may be because minimal EIMD or increase in strength is induced by a step-down protocol, attenuating 29 the magnitude of cross-education. However, this questions whether unilateral RET will induce CL-RBE, despite strength transfers (183). While several mechanisms have been linked to RBE, given the absence of direct mechanical exposure, contralateral-RBE (CL-RBE) is likely mediated predominantly by neural mechanisms and possibly by adaptations to the inflammatory response (4,184). CL-RBE can arise from different sites of the nervous system. The current conjecture for neural modulation follows two frameworks (185). “Cross-activation” models encapsulate the excitation of contralateral cortical motor areas during unilateral execution of movements (186). Alternatively, “bilateral access” models postulate bilateral access of neural circuits generated by unilateral exposure (187). Indeed, cross-education studies reveal greater cortical motor response (188) and increased muscle fiber activation in the CL limb (189). Upstream neural adaptations can impact acute inflammation adaptations, for example, attenuated inflammatory modulatory regulator, nuclear-factor kappa β, following the second bout of ECC contractions in the CL limb (190). Increased recruitment of slow-twitch motor units rather than damage susceptible fast-twitch likely mediates less initial EIMD and reduced inflammation response hence decreased nuclear-factor kappa β. Some studies have postulated the release of circulatory endocrine factors as a mechanism for CL-RBE (75,191,192). If this is the case, RBE would be observed in remote unrelated muscle groups. However, RBE was not detected in the elbow flexors when prior lower limb, knee flexion and extension, ECC exercises were performed, and vice versa; no protection was conferred by maximal elbow flexor ECC to later knee flexion ECC (172,184). Results from both studies concur that circulating factors are unlikely candidates in CL-RBE. Furthermore, no CL-ECM adaptations have been reported, therefore, it is to be assumed that ECM molecular 30 signatures are confined to local remodeling. Even if muscle-derived molecules circulate, their effects will be minute compared to the direct application of exercise on the muscle. Adaptations as a result of resistance exercise are a sum of ‘morphological adaptations and neurophysiological activation’ and the lack of phenotypic alterations (i.e., hypertrophy) abate the influential properties of cross-education, at least in attempts to quantify local changes. 1.7 Summary A maximal ECC bout is classically administered in RBE literature, a phenomenon reflective of acute adaptation to RET especially in the naïve muscle. Some studies have deviated from the traditional focus on high force contractions to demonstrate, albeit to a lesser extent, the ability of submaximal contractions in conference of protection. Single bouts of electrical stimulation, ISO, CON, and 10-40% ECC contractions, as well as repeated submaximal ECC exercises, have been used to successfully attenuate damage when performed prior to the maximal ECC bout. Yet, the ability of RET, comprised of previously tested preconditioning modalities, has not been studied as a stimulus of RBE. RET is known to induce gene expression profiles indicative of ECM remodeling, a mechanism known to be involved in RBE (4,193). Given the similarities in adaptation, it is of interest to contrast RET to one maximal ECC bout in their magnitude of conferred protection and involvement of ECM remodeling. 1.8 Purpose The primary purpose of this thesis was to determine whether RET can attenuate strength loss following 300 ECC contractions to induce RBE and examine acute and prolonged ECM-related changes following resistance exercise and repeated ECC bouts in young healthy adults. 31 1.9 Hypothesis Primary: • The trained leg will demonstrate a smaller decrease in strength following B1 compared to the control leg at 24h, and 48h post B1 • The trained leg will demonstrate a smaller decrease in strength following B2 compared to the control leg at 24h, and 48h post B2 • The trained leg will have higher COL1A1, COL3A1 and COL4A1 gene expression pre-B2 (0h) compared to the control leg • The trained leg will have higher COL1A1, COL3A1 and COL4A1 gene expression post-B2 (0h) compared to the control leg Secondary: • The trained leg will have higher MMP2 gene expression at post-B1 (48h) compared to the control leg • The trained leg will have lower MMP9 gene expression at post-B1 (48h) compared to the control leg • The trained leg will have higher TGFβ gene and protein level at pre-B1 (0h) compared to the control leg • The trained leg will have lower TGFβ gene and protein level at pre-B2 (0h) compared to the control leg Tertiary: • The trained leg will have lower TN gene expression at post-B1 (48h) compared to the control leg • The trained leg will have lower FN gene expression at post-B1 (48h) compared to the control leg 32 Chapter 2: METHODS 2.1 Participants 14 healthy (i.e., free of diabetes, cardiovascular disease, or any medical conditions that impact ability to undertake strenuous physical activity) adults volunteered to participate in this study, 6 Females and 8 Males (20.94 4.14 years) (Table 1). Verbal and written informed consent was obtained from all participants after they were informed of all procedures and potential risks. The participant's habitual activity ranged from sedentary to recreationally active, but none was actively training in any particular sport nor had they participated in lower or upper body strength-training programs for at least 6 months prior to inclusion in the study. Sample size was calculated based on the primary endpoint of the percentage of strength loss using MVC with the primary time point of three days following the first damaging exercise bout using G*Power (Version 3.1.9.4.). It was assumed that RET would attenuate force loss by approximately 80% as much as a single bout of damaging ECC exercise and that employing the within-subject unilateral model would result in a between leg correlation of 0.8 as suggested by MacInnis and colleagues (178). Based on these assumptions and a 12% (σ = 18%) repeated bout effect as reported by Hyldahl and colleagues, a sample size of 14 was calculated (4). All participants agreed to refrain from participating in new physical activity or take oral or topical analgesics for the duration of the study. None of the participants had previous biopsies in the VL. Participation in the study was completed between October 2021 to February 2022. This study was approved by the Research Ethics Board of University of British Columbia (H21-01924) and all procedures conformed to the Declaration of Helsinki. 33 Table 1. Participant baseline characteristics 2.2 Randomization Participants had one leg randomly assigned to be in the trained condition and one leg randomly assigned to be in the control condition. The dominant leg was identified and randomized to each condition. Limb randomization for each participant was completed using an online coin flip, with heads designating the right leg for training and tails the left. The other leg was assigned as the control leg. In total, there were 14 participants in each condition, trained and control, as the sample size will be 14 and both conditions are assigned to each participant. Each participant was assigned a study identification number that corresponded to the time at which they enrolled. 2.3 Study Overview An overview of the study timeline is illustrated in Figure 4. The total duration of the study was 5-6 weeks and consisted of 6 unilateral RE training sessions and 2 maximal ECC bouts. RE sessions took place over 2-3 weeks with each session interspersed by at least 1 d, and the last session taking place at least 2 d prior to the first biopsy. All RE sessions was only N = 14 Age (yr) 20.9±4.14 Height (m) 1.74±0.11 Weight (kg) 71.7±15.6 BMI (kg/m2) 21.5±7.2 Values are means ± standard deviation. BMI, body mass index. 34 performed by the designated trained leg. All training sessions were conducted in the Allan McGavin Sports Medicine Clinic at the Chan Gunn Pavilion under the supervision of at least one member of the research team. Muscle biopsies occurred in both the Allan McGavin Sports Medicine Clinic and Medical Science Block C, Room 108 Muscle function testing conducted on the Biodex was obtained at the UBC Hospital, Rm 326. Following the completion of all 6 training sessions, 2 ECC bouts were performed separated by 21 d. 8 biopsies were taken in total with 2 bilateral biopsies obtained on each designated biopsy day, immediately prior to ECC B1 (designated as pre-B1), 48 h after (post-B1), and right before (pre-B2) and 48 h ECC B2 (post-B2). Strength assessment was conducted prior to and 24, 48, 72h the first training session, B1 and B2. Figure 4. Schematic of Study Design Control Trained 35 2.4 Initial Contact After the participant contacted the associate investigator about potentially enrolling in the study, the participant information sheet was sent by email outlining the details of the study. The email included a brief overview of the study design and procedures, the inclusion/exclusion criteria, as well as answered any preliminary questions the participant had. If the participant was interested in enrolling in the study the associate investigator set up a time to complete screening/familiarization with the participant. 2.5 Screening/Familiarization At least one week prior to the commencement of RET, the prospective participant visited the lab for screening and familiarization. This included an explanation of the study procedures and risks, followed by the provision of written consent to participate. Screening for the study also included the completion of The Canadian Society for Exercise Physiology Get Active questionnaire (CSEP-GAQ) which was used to determine physical activity and readiness for exercise (194). This questionnaire also determined whether participants are able to safely perform our exercise protocol. Additionally, if a participant answered ‘yes’ to any question on the CSEP-GAQ, they will be given the CSEP-GAQ reference document for their own personal use on whether or not to get in contact with their health care provider prior to engaging in more physical activity. Finally, the participant was introduced to the exercise protocol which included seated unilateral leg extensions and leg press as well as a 1RM test using the designated trained leg on the leg press and leg extension machine for determination of future training loads. Prior to testing, participants were asked to warm-up on a stationary bike for 5 min. Thereafter, the 1RM was obtained first on the leg press then on the leg extension, as this was the order of exercise on training days. 36 2.6 Muscle Function Assessment This assessment for MVC was performed immediately prior to the first RE session for baseline MVC values of both legs, and 24h, 48h and 72h after the first RE session, B1 and B2. A 5-minute warm-up was first completed on a stationary bike at a self-determined RPE of 8 on the Modified Borg scale. Then, participants were seated at 90° hip angle with the lateral epicondyle aligned with the axis of the dynamometer and provided an explanation on the Biodex dynamometer. The lower limb was fixed to the chair 2 cm above the malleolus. A hip and chest strap fixed the individual into the chair. Positioning of the seat and lever arm was documented and remained identical throughout the study. Full knee extension (0°) and flexion (90°) was entered as a reference value on the dynamometer system. Prior to the start of the MVC trials, the participants completed 2 warm-up sets of isometric contractions, first at 50% of effort and the second at 75%. A 30 second rest was given between the two trials. Three (5 s) trials at 70° of knee flexion with 30 s rest between trials were completed and the isometric average torque values were defined as the mean of the highest attainable value among the three trials. During each MVC attempt, the researcher provided a verbal countdown and strong encouragement throughout each contraction. 2.7 Resistance Exercise Training In total 6 RE session was completed on the assigned trained leg. Each participant performed 3 sets of unilateral leg press and knee extensions (Atlantis Strength, Canada), in that order, until volitional fatigue was reached within 10-12 repetitions. The ECC portion of the movement was completed in 3s as guided by the investigator. For leg press, the participants were instructed to push up (concentric) in 1s and lower for 3 s (eccentric), as counted by “up, 1, 2, 3…”. For the leg extensions, the participant was instructed to kick from the flexion position 37 (~90) to extended (~180), using the same counting method. The initial load was determined using the 1RM value obtained from the familiarization session. For leg press and leg extension, the load was set as 80% and 70% 1RM, respectively. Since leg press was the first exercise performed, participants were often fatigued by the second exercise and could not complete the first set within the rep range unless the load was lowered to 70% 1RM. 90 seconds of rest was provided between each set and 120 seconds between each exercise. The training load was adjusted to reach volitional fatigue as the participant got stronger. When a participant surpasses the target repetition range during either of the first two sets, their load will be increased by 10 lbs during the next set. Similarly, if the number of repetitions performed did not reach the minimum, 10, the weight will be dropped for the next set. During the last set, if the number of repetitions reached is at or above 12, the load was increased during the next training session. On the last day of RET, the participant was given printed copies of food logs and instructed to record and consume a similar diet 24 h prior to each muscle biopsy. They were also instructed to complete an overnight fast the night prior to the muscle biopsy. 2.8 Muscle Biopsy 4 bilateral biopsies, 8 biopsies in total, were collected immediately prior to ECC B1 and B2, as well as 48h after. Participants were asked to overnight fast prior to the biopsies. Under local anesthesia (2% lidocaine), a small incision was made into the skin and fascia, and the Bergström biopsy needle modified for manual suction was inserted into the muscle. Manual suction was used to withdraw 25 - 200 mg of muscle. Muscle samples were divided into sections for protein and mRNA homogenates, and microscopic analysis, and immediately frozen in liquid nitrogen at -80°. Tissue portions designated for microscopic analysis were mounted on a cut pipette tip with optimal cutting temperature compound (OCT) and frozen in isopentane cooled 38 by liquid nitrogen. All biopsies were performed between 7:00 AM and 12:00 PM, local standard time, except for one biopsy that was conducted at 4pm due to scheduling conflicts. 2.9 Eccentric Bout Two ECC bouts were completed with a dynamometer (Biodex Medical Systems), one 2-3 weeks after the initial RE session (denoted as B1) and the other 21 d after (B2). A familiarization bout was performed prior to the first RE session after MVC data was gathered with the same chair configuration as that of MVC, consisting of 5 reps. The leg that experienced the familiarization was alternated each time. After the training period in the trained leg, participants completed a bilateral ECC bout consisting of 300 repetitions as 30 sets of 10 contractions. Each set was interspersed by 60 seconds of rest. The order of the leg was alternated. Participants were asked to resist the dynamometer as it moves from 30 to 110 at an angular velocity of 120/s. During the session, the participant was asked to exert maximal force. After the protocol, the participants were advised to avoid strenuous exercise or any pain mediation until the last muscle function assessment was completed at 72h post-ECC. Total work was recorded during both bouts. Following the biopsy, the participants were provided with food (i.e., granola bars, instant noodles) if they chose to eat prior to the ECC bout. 2.10 Sample Analysis 2.10.1 Area Under the Curve for Isometric Force Loss Area under the curve (AUC) was the sum of the area under each time point MVC was obtained (0h, 24h, 48h, 72h) following RET, B1 and B2. To determine the magnitude of protection conferred by RET, force loss in the trained and control leg following B1 and B2 was 39 compared. The average force loss between both legs post-B1 and Post-B2 was calculated to determine protection conferred by B1 to B2. 2.10.2 TGFβ Protein The frozen muscle samples (~30-70mg) were first homogenized in a TissueLyser bead mixer (Quagen, CA, USA) with a radioimmunoprecipitation assay buffer (RIPA buffer: 50 mM Tris at pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 1% deoxycholate with protease and phosphatase inhibitor tablet (Thermo Scientific, Rockford, IL) then centrifuged at 700g for 15 min. The supernatant was collected and total protein concentration was determined by Multiskan SkyHigh plate reader (Thermo Scientific, Rockford, IL). Active TGFβ quantities in muscle lysates were measured using commercially available ELISA kit: Quantikine Human TGF-beta1 Immunoassay (R&D Systems). Samples were first acid activated, then diluted with the provided calibrator diluent (Calibrator Diluent RD6-11). 75ug of protein was used per well for each sample. All samples were ran in duplicates and the value for each sample was the mean of the duplicates. Optimal density of each plate was read with a Multiskan SkyHigh plate reader (Thermo Scientific, Rockford, IL)The optimal density of each well were measured at 450nm, and readings at 540nm was subtracted to correct for any optimal imperfections. Concentrations (pg/mL) were determined from the standard curve of each plate after subtracting the blank and multiplying the optimal density read off for a dilution factor of 1.4 to correct for dilution of each sample with the calibrator. Amount TGFβ protein (pg) per mg of wet muscle was calculated by determining the weight of muscle in lysate added per well, then dividing the value by protein concentration read off the standard curve. 40 2.10.3 RT-qPCR Total RNA was isolated from 20-40 mg of frozen muscle by Trizol reagent (Ivitrogen) according to the manufacturer’s recommendations and was isolated using the RNeasy mini kit (Qiagen, CA, USA). Two micrograms of RNA were reverse transcribed by use of a master mix consisting of random primers, buffer, dNTPs, and reverse transcriptase from Applied Biosystems kit (#43-688-14). For each target mRNA, 0.2 μl cDNA was amplified in a 5 μl SYBR Green PCR reaction containing 1 × Quantitect SYBR Green Master Mix (Qiagen, CA, USA). Amplification was performed using PCR thermal cycler (Applied Biosystems). The Geomean of GAPDH and VCP was used as an internal control. Data was the mean of triplicate Cq values and values for the trained leg were expressed related to the first biopsy of the controlled leg using the Cq log normalized method. The primer sequences used are depicted in Table 2. Table 2. Primer Sequences Gene Forward 5'- 3' Reverse 5'- 3' COL1A1 TTCTGTACGCAGGTGATTGG GACATGTTCAGCTTTGTGGA COL3A1 CACGGAAACACTGGTGGACAGATT ATGCCAGCTGCACATCAAGGAC COL4A1 GAGGAAAGAGGTGAGAAAGAAGG CCTGATGAAGGCATAAGGAGTAG TGFB CCCAGCATCTGCAAAGCTC GTCAATGTACAGCTGCCGCA MMP2 AGATCTTCTTCTTCAAGGACCGGTT GGCTGGTCAGTGGCTTGGGGTA FN CCACCCCCATAAGGCATAGG GTAGGGGTCAAAGCACGAGTCATC TN ACCATGCTGAGATAGATGTTCCAAA CTTGACAGCAGAAACACCAATCC 41 2.11 Statistical Analysis Data analyses were carried out using the Statistical Package for the Social Sciences (version 12.0.1; SPSS, Chicago, IL) and all data are expressed as means ± standard deviation (SD). Three-way repeated measures ANOVA was applied to analyze MVC data with factors time (0h, 24h, 48h, 72h), bout (RE, B1, B2) and condition (trained leg vs. control leg) for all participants. The same three-factor ANOVA was used for gene and protein data, time (0h vs 48h), bout (B1 vs. B2) and condition (trained leg vs. control leg). Differences were considered statistically significant at P < 0.05. Cohen’s d effect size (ES) was calculated for post-hoc analysis, and the threshold values were ES > 0.20, ES > 0.50, ES > 0.8, and ES > 1.2. Area under the curve (AUC) was used to determine the magnitude of protection conferred by RET to ECC B1. Two-way ANOVA was conducted for work completed between B1 and B2 with factors condition (trained vs. control) and Bout (B1 vs. B2). All data was assessed for normality using the Shapiro-Wilks test. Extreme outliers were removed prior to any analysis and were identified by the SPSS software as being data points greater than three interquartile ranges from the end(s) of the boxplot(s). 42 Chapter 3: Results 3.1 Exercise Performance and Muscle Function Participants performed 6 sessions of RE on the designated trained leg and 2 bilateral 300 ECC bouts on both legs separated by 21 days. There was no difference (Bout x Condition, p=0.235) in the amount of total work completed in B1 between the trained leg (81564153J) and the control leg (64363652J) or in B2, between the trained leg (73493826J) and control leg (72284132J). EIMD was assessed as strength loss by measuring MVC. RET significantly protected against strength loss from B1 in the trained leg by 42% using area under the curve (p=0.03) (Figure. 7). AUC for both legs also showed protection of 40% from B1 to B2 (p<0.001). In both legs, 300 ECC contractions induced force loss at 24h and 48h, and strength returned to baseline by 72h in B1 (Time, p=0.005) (Figure. 5). Both ECC bouts induced greater damage compared to the first RE bout (Bout, p<0.001). Strength loss was only significant at 24h Post-B2, compared to at both 24h and 48 Post-B1 (Time x Bout, p=0.011). Three-way ANOVA analysis indicated no strength loss in either leg following the first RE bout, B1 or B2. After RE force loss compared to baseline (0h) was at 24h (2.914.09%), 48h (2.914.09%), and 72h (3.914.1%) in trained leg or 24h (1.613%), 48h (2.513.3%), and 72h (2.314.9%) in the control leg. Following B1, at 24h (13.514.3%), 48h (16.814.8%), and 72h (9.117.6%) in the trained leg, and at 24h (23.421.5%), 48h (28.823.1%), and 72h (18.324.9%) in the control leg. Decreases in MVC post-B2 were in the trained leg at 24h (15.516.8%), 48h (0%), and 72h (0%) or in the control leg 24h (19.319.2%), 48h (0%), and 72h (0%). 43 Control Trained0510152025Area Under the Curve (AU)sControl Trained051015Area Under the Curve (AU)ControlTrainedA BFigure 6. Summative protection conferred by A) RET B1 B) from B1 to B2. Values are expressed as meanSD σP < 0.05 significance relative to control. Figure 4. Summative protection conferred by A) RET B1 B) from B1 to B2 Values are expressed as an meanSD σP < 0.05 significance relative to control. Figure 5. Indirect measures of muscle damage following 2 bouts of 300 ECC contractions. Changes in maximal voluntary knee-extension torque 3 d post-RE –B1 and –B2. Values are expressed as an meanSD ▲P < 0.05 significance relative to baseline (0 d). ▽ P <0.05 significance relative to 2d post-B2 (48 h). 44 3.2 ECM-related Gene expression ECM transcriptional regulation was measured by qPCR to quantify ECM remodeling following RET and damage. Transcripts corresponding to collagen synthesis, degradation, and remodeling were measured. The expression of COL1A1 (Bout, p=0.020), COL3A1 (Bout, p=0.014) and COL4A1 (Bout, p=0.046) increased significantly pre- and post- B2 compared to B1 (Figure. 7). Only COL4A1 in the trained leg was upregulated after 300 ECC contractions (Time × Condition, p=0.027) (Figure. 7). ECC B1 increased COL1A1 (Time x Bout, p=0.045) in both legs. MMP2 mRNA levels were significantly higher in B2 (Bout, p=0.002) and levels increased postexercise in the control leg (Time x Condition, p=0.049) (Figure. 8). Of the transitional matrix genes, only TN was significantly upregulated for both legs after B1 and B2 (Time, p>0.001) at 48 h. B2 also resulted in higher levels of TN (Bout, p=0.007). No change was seen in FN (Table 3; Figure. 9). 45 0h 48h 0h 48h 0200400600COL1A1 mRNA relative to Control Pre-B1B1 B2 ♐0h 48h 0h 48h 0246810COL3A1 mRNA relative to Control Pre-B1 B2B10h 48h 0h 48h 0510152025COL4A1 mRNA relative to Control Pre-B1 B1 B2 BControlTrainedACFigure 7. mRNA expression of A) COL1A1, B) COL3A1, and C) COL4A1 measured pre- (0 h) and post- ECC B1 and B2. Data are displayed as fold change relative to pre-B1 (0 h) sample from the control leg and means are ± sd. *P < 0.05 significance relative B1. †P < 0.05 interaction effect between time x condition relative to baseline (0 d). ♐︎P<0.05 interaction effect between time x bout relative to baseline (0 d) 46 0h 48h 0h 48h 051015MMP2 mRNA relative to Control Pre-B1 B1 B2 ControlTrainedFigure 9. mRNA expression of A) FN and B) TN measured pre- (0 h) and post- ECC B1 and B2. Data are displayed as fold change relative to pre-B1 (0 h) sample from the control leg and means are ± sd. #P < 0.05 significance relative to pre-B1 and B2 (0 h). Figure 8. mRNA expression of MMP2 measured pre- (0 h) and post- ECC B1 and B2. Data are displayed as fold change relative to pre-B1 (0 h) sample from the control leg and means are ± sd. *P < 0.05 significance relative B1. †P < 0.05 significance relative to pre-B1 (0 h).9 47 3.3 Transforming Growth Factor Beta At the gene level, TGFβ is significantly upregulated at B2 (Bout, p=0.009), in the control leg, TGFβ increases post-ECC (Time x condition, p=0.018; Table 3). ELISA analysis of muscle lysate showed no significance of protein level changes (Figure 10). 0h 48h 0h 48h 051015TGF-b1 mRNA relative to Control Pre-B1 B1 B2 A B0h 48h 0h 48h 0246810TimeTGFb relative to wet muscle (pg/mg)ACBDB1 B2Figure 10. A) mRNA expression of TGFβ, measure pre- and post- ECC B1 and B2. Data are displayed as fold change relative to pre-B1 (0 h) sample from the control leg and means are ± sd. *P < 0.05 main effect of bout relative B1. †P < 0.05 interaction effect between time x bout relative to baseline (0 d) B) TGFβ1 protein in skeletal muscle following 2 bouts of 300 ECC contractions assessed by a quantikine ELISA immunoassay. Shown are the protein (pg) per weight of wet muscle (mg). Data are means ± sd. 48 Table 3. Summary of Gene 3-Way ANOVA Interactions. Bolded p-values indicate significance of p < 0.05. GENE TIME BOUT CONDITION TIME × BOUT TIME × CONDITION BOUT × CONDITION TIME × BOUT × CONDITION COL1A1 P=0.658 P=0.020 P=0.940 P=0.021 P=0.685 P=0.425 P=0.067 COL3A1 P=0.929 P=0.014 P=0.218 P=0.707 P=0.536 P=0.313 P=0.389 COL4A1 P=0.189 P=0.046 P=0.642 P=0.267 P=0.047 P=0.952 P=0.398 TN P<0.001 P=0.007 P=0.511 P=0.700 P=0.503 P=0.219 P=0.955 FN P=0.202 P=0.327 P=0.595 P=0.252 P=0.986 P=0.159 P=0.778 TGFΒ P=0.187 P=0.009 P=0.610 P=0.941 P=0.022 P=0.630 P=0.270 MMP2 P=0.975 P =0.002 P=0.813 P=0.306 P=0.013 P=0.440 P=0.299 49 Chapter 4: Discussion & Conclusion 4.1 Main Findings The aim of this study was to determine whether short-term RET can confer protection against EIMD following 300 ECC contractions and elucidate ECM-related mechanisms that may contribute to acute adaptations to RE in healthy young adults. Several gene candidates involved in degradation (MMP2), collagens (COL1A1, COL3A1, COL4A1), promotion of collagen synthesis (TGFβ) and regeneration (TN, FN) were selected. The results demonstrated that (i) RET protected against strength loss, a marker of EIMD, following ECC B1 (ii) 300 ECC contraction induced RBE to ECC B2 (iii) acute and prolonged ECM reconstructive activity may be a mechanism of RBE and responsive to RET 4.1.1 Maximal ECC Contractions and RET as a Stimulus for the Repeated Bout Effect RET in addition to previously used preconditioning exercises mitigated strength loss following damaging ECC contractions (39,43,45). 6 sessions of RE were successful in attenuating ECC-induced EIMD by 42% compared to the control leg (Figure 8). The majority of previous reports of successful preconditioning exercises were of the elbow flexors. Chen and colleagues (44,45) showed that 30 submaximal ECC contractions set to a load 40% of an individual’s MVC when performed once, two weeks prior to a maximal ECC bout attenuated damage markers, including force loss and DOMS, by 20-60%. When 30 submaximal ECC contractions were performed 4 times every 2 weeks, it reduced damage markers by 60-100%. 2 and 10 ISO contractions Similarly, ISO and ECC contractions set to a load of 20% MVC and 2 or 10 ISO-MVC conferred protection when 30 maximal ECC contractions were performed 2 d and 3 weeks later, respectively. (39,43). Potential protection from submaximal exercise in the 50 knee flexor has been less researched, with only one study showing that 3 sets of 10 knee extensions on standard exercise machines attenuated EIMD markers when 30 maximal ECC was performed 2 or 7 days later. No previous study has investigated recovery in MVC of the knee flexors following RET as a preconditioning exercise. Strength decrements in the trained leg Post-B1 at 48h (16.8%), and 72h (9.1%) were similar to, and lower than post-B2 values observed by the Hydahl group at 48h (19%), and 72h (19%) (4). It is apparent that 6 sessions of RE mirror that of 300 ECC contractions in the magnitude of protection conferred. Induction of RBE is suggestive that RET may have similar remodeling kinetics as that of an acute damaging bout. The RBE paradigm is interesting as a single bout of exercise stimulates sufficient adaptation to prime the muscle and its ECM so that less damage occurs when a subsequent maximal ECC bout is performed. Protection conferred by RET suggests that EIMD is not necessary for RBE and summation of adaptations from a short period of training can protect against a similar and more damaging exercise type. The exact duration of protection conferred by a preconditioning exercise is unknown. Lin Mu Jin and colleagues using 30 low intensity ECC contractions of the knee flexors reported 30-66% protection from 30 maximal ECC contractions was eliminated between 7-14 d after completion of the preconditioning exercise and protection was abolished at 14 d (40). Prevalent protection seen in the current study 2-3 d after cessation of training is consistent with this time frame. However, since the damaging bout used in the current study consisted of 270 more repetitions compared to 30, a greater magnitude of protection was likely required of the preconditioning exercise. If protection from 30 low intensity ECC contractions lasts up to 1 week, protection from subsequent RE bouts may be additive if performed before the protective effects of the previous RE bout have dissipated (Figure 11). Consecutive addition by repeated 51 RE sessions likely increases magnitude of protection after 6 sessions, providing an explanation for how RET can mirror one maximal ECC bout. Similar to previous reports, maximal ECC exhibited RBE; no isometric force loss was present at 48h post-B2 compared to significant damage experienced at both 24h and 48h post-B1, indicating protection as well as accelerated recovery (4,39,85). Although decreases in MVC were not significant relative to baseline, percentage of strength loss following the first bout of 300 ECC contractions in the control leg at 48h (28.8%) and 72h (18.3%) were in parallel with results reported by Hydahl and colleagues, at 48h (24%) and 72h (33%), albeit with a slightly earlier peak strength loss in the present study (4). Post-B2, the present study exhibited 100% protection at 48h and 72h from 300 maximal ECC contractions, whereas the Hyldahl group reported 21% and 42% at 48h and 72h, respectively (4). Despite an identical damage protocol, the magnitude of protection was greater. Similar total work was completed in both bouts yet B2 failed to induce any strength loss at 48h or 72h, in either leg. Several reasons may have contributed to the elimination of strength impairments. Greater residual protection may have been present during the timepoint B2 was completed in the current study. Since conferred protection is diminished over time (39), at 21 days, the effects of B1 are likely more potent than at 28 days, the rest period implemented between bouts by the Hydahl group (4). Cross-education from the RET limb may explain the accelerated recovery where peak strength loss occurs at 24 h instead of 48 h as in the Hyldahl study (4), as well as absence of damage post-B2. If adaptation to RET summates, CL-RBE likely also summates albeit to a lesser degree. While in the trained leg, conferred protection after 6 sessions reach a level similar to that of a maximal ECC bout, in the control leg, probably only a fraction of protection is crossed-over. 52 There has only been one study investigating the effects of CL-RBE of the knee flexors. CL-RBE by 60 maximal ECC contraction was evident at 7 d, but lost at 28d (195). The number of contractions performed influences the magnitude of attenuation (28). 6 maximal ECC contraction was more effective than 2 in attenuating EIMD following 24 maximal ECC contractions (196). There is a lack of empirical studies on whether a greater number of contractions can increase the duration of conferred protection. Protection from 300 maximal ECC contractions likely results in a greater initial magnitude that takes longer to dissipate. Drawing on strength transfer studies, since ~22% of total strength is gained on the CL limb from the trained limb, mediated largely by neural adaptations, (180), a similar gain in percentage of protection likely occurred, accelerating recovery post-B1, and persisted into B2 for the complete recovery of force loss by 48 h (Figure 12). Mechanistically, CL-RBE is likely a result of neural adaptations such as altering motor unit recruitment or firing. These modulations primes the muscle so that it is more efficient, incurring less damage. Other than nervous system influences, global molecular changes may play a role. Unilateral electrical stimulation of rat soleus muscle revealed 66 affected genes in both the stimulated and CL-limb, mostly stress-response genes, including HSPs (197). HSPs contribute to the inflammatory response and have been shown to be attenuated by repeated bouts. Activation in the CL-limb may alter or reduce inflammation postexercise. Studies attempting to elucidate the underlying mechanism of cross-education in limb immobilization hypothesize release of circulating endocrine factors (i.e., insulin growth factor 1) (198,199), and others such as interleukines (200), ROS (201), myostatin to list a few (202), from the trained limb in prevention of atrophy in the CL-Limb. Accelerated recovery may simply have been a factor of less MVC loss. A meta-analysis concluded that greater MVC loss at 24 to 48 h or severe EIMD resulted in slower MVC torque 53 recovery time (203). The lower level of EIMD induced by B2 may not have required a longer recovery period. Bilateral execution of ECC used in the current study as compared to an unilateral approach employed by the Hydahl group may have increased protection from the CL limb (4). Given that 300 ECC contractions were performed, it is likely that CL-RBE from both RET and B1 did not dissipate and contributed to greater protection following B2, resolving damage in both legs. A familiarization session was not performed prior to the collection of baseline MVC. Thus, we cannot dismiss that baseline MVCs were likely lower than true values considering a learning effect and increases in strength due to RET sessions. Isometric data demonstrate that the reliability of session one data is only moderate-to-high, which increases to high by session 2 (204). A familiarization prior to obtainment of baseline values may demonstrate greater damage post-ECC. 54 4.1.2 ECM Remodeling in early adaptation to RET The adaptation of the ECM to exercise is a highly dynamic process where quantitative and qualitative changes can alter the skeletal muscle architecture (207). It has been postulated the time course of ECM remodeling is associated with functional outcomes, such as resistance to damage (3,4). Although the present study was only statistically powered to detect strength loss, ECM related transcriptional changes still provides clues into remodeling following acute exercise and training. ECM remodeling likely follows a biphasic structure, sequenced as first catabolic then anabolic (23). Our data generally trends towards this time course, as seen by a delayed upregulation of anabolic genes COL1A1, COL3A1, COL4A1, and TGFB. Expression of these genes are not changed by acute exercise but rather demonstrate prolonged elevated transcriptional activity with repeated exercise. Although MMP2 can be categorized as catabolic, Figure 11. Schematic of conferred protection by RET and maximal ECC contractions. 55 its diverse role, working to liberate TGFB bound to LAP and facilitating movement of MuSC, may explain its later appearance. Notwithstanding, hypertrophy only occurs after resolution of the repair phase at around the 3 week mark (12), it is plausible that ECM cues aid this process adjacent or thereafter to this period. 4.1.2.1 Delayed Increase of Profibrotic Genes Following Maximal Eccentric Contractions The collagen superfamily are the main components of the ECM and are known to be responsive to exercise. RE induce rapid secretion of collagen genes which halts until the initial repair phase terminates (98). With training, COL1A1, COL3A1 as well as COL4A1, are continuously elevated (207). Results presented here supports delayed upregulation, mainly by activity of COL1A1, COL3A1 and COL4A1. All measured collagen genes increased 3 weeks after the initial damaging bout, the same pattern of expression previously reported (4). In rats, collagen type IV concentration was only elevated in the damaged white part of the quadricep muscle, with no change detected in undamaged muscle after downhill running. The authors suggested that collagen type IV concentration was most likely inversely correlated with muscle damage (101). If this inverse relationship translate to humans, greater damage as measured by greater strength loss post-B1 may explain absence of COL4A1 and its appearance post-B2 when force loss was attenuated. HIIT and RE has been shown to induce an increase in the expression of COL4A1 immediately postexercise (17,193), but HIIT and RE generally does not result in the same level of damage as maximal ECC contractions. Damage following maximal ECC contractions may delay induction of COL4A1. Upregulation of COL4A1 post-B1 and -B2 in the train leg likely reflects this. RET may have induce sufficient adaptations so the attenuation of damage allows for the increase in expression. Similarly, higher expression of COL4A1 in both 56 leg post-B2, may be a result of attenuation conferred by B1. Considering the composition of the basement membrane is primarily collagen type IV, initial downregulation when damage is apparent likely allows movement of molecules important for clearance of debris and MuSC for regeneration. Subsequent upregulation once damage subsides, perhaps point to repair and restoration of the basement membrane. Upregulated COL1A1 48 h post-B1 is in contrast with previously reported data where a single bout of ECC contractions did not upregulate COL1A1 at either 3h or 48 h postexercise (4). Only activated MuSC display higher levels of COL1A1 gene expression, suggesting adaptations conferred by RET may have mitigated damage induced by ECC contractions to a degree that bypass the repair phase to allow for MuSC activation (208). Future studies should correlate collagen gene expression with protein levels either by assessing fractional synthesis rates or collagen staining of the muscle tissue. Somewhat in contrast, collagen type IV concentration was reduced concomitantly with TIMP-2 1 and 2 d after exercise in the red part of the rat soleus (101). Reduce TIMP-2 inhibition capacity may increase MMP2 degradation degradative capacity. In the present study, at the transcriptional level, only post-B2 in the control leg was the rise in COL4A1 concomitant with a decrease in MMP2. Whereas, in the trained leg, MMP2 and COL4A1 were both upregulated post-B2. This may be because training allows for rapid repair where degradation and synthesis of collagen type IV occur simultaneously. The TGFβ pathway appears to be central for the regulation of collagen type I and III expression, and fibroblast transdifferentiation to myofibroblast in the skeletal muscle. It is also a chemoattractant of fibroblast to the site of injury as a primary source of ECM components (209). TFGβ is secreted in its latent form bound to LAP and must be liberated from LAP in order to bind to its receptor, TGFβII. Mechanical strain and damage appear to increase TGFβ mRNA 57 in rats (210). In the present study, 300 ECC contractions and the damage that ensued did not upregulate TGFβ mRNA levels until 21 d afterwards. This upregulation aligns with increased transcription of collagen genes pre- and post-B2. In comparison to the trained leg, TGFβ decreased in the control leg after B1 and B2. This may be an effect of greater damage after exercise. Overproduction of TGFβ drives a fibrotic phenotype, therefore, downregulation may be a mechanism to decrease collagen production and prevent excessive fibrosis during healing. Another possibility is the transient upregulation of TGFβ genes between 0 to 48h after exercise, prior to the biopsy timepoint of this study. In contrast, no change was seen in active TGFβ protein levels before or after exercise. RE has been shown to elevate circulating latent TGFβ at 2 week and 3 week postexercise (211). Muscle TGFβ may have been released into circulation before activation. TGFβ is implicated in an array of processes, for instance, remodeling of the bone or immune functions (212,213). Mechanical strain may increase secretion external to the localized area to act on systemic targets, for instance Another explanation could be that active TGFβ once released from LAP is rapidly internalized by binding to its receptors. Downstream transcriptional factors have been shown to increase in expression rapidly after exercise (214). Or it is also possible that activation of TGFβ is delayed as suggested by in vitro and in vivo studies where grown injured myotubes displayed increased total TGFβ concentration without change in active levels (215). Immediate injection of antifibrotic agents also did not affect muscle healing (216). Muscle strain injury in rats produced by stretching of the muscle revealed the presence of latent-TGFβ by immunohistochemistry in areas of myofiber injury 48 h after, with no change in active TGFβ. This supports the increase of the latent product and rapid consumption of active TGFβ by its receptor. An increase in TGFβRII protein, the primary receptor of TGFβ was seen 27 d after 300 ECC contractions with 58 levels remaining elevated 2 d after ECC B2 reported by the Hyldahl Group (4). Response of the receptor aligns with TGFβ gene expression, and the absence of change at the protein level likely indicates an increase of latent TGFβ. Additional studies are necessary to understand the mechanism driving activation of TGFβ and its regulation, perhaps by investigating levels of latent TGFβ in the muscle postexercise. 4.1.2.2 Transitional Matrix Following damage, upregulation of a transitional matrix consisting of TN, FN, and Hylaronic acid appears to provide instructional cues for cell behaviours (141). TN is thought to respond to mechanical stimuli to promote early deadhesion in the skeletal muscle. TN was upregulated 48h post-B1 and -B2 in both legs. This coincides with previously reported transient upregulation in immunoreactivity after maximal ECC contractions (4). Contrarily, upregulation in TN gene expression post-B2 does not align with immunoreactivity results following a repeated bout of 300 maximal ECC contractions where TN immunoreactivity was attenuated by the second ECC bout when 4 high responders were excluded (4). Slow turnover rates of ECM protein could explain the discrepancy between elevated gene expression and attenuated protein response following B2 (159). It could also be that mechanical stress always increases mRNA levels, but demands of the muscle regulate translation. Appearance of TN is limited to sites of active remodeling, acting antagonistically to the adhesive properties of matrix proteins such as FN (161). Antagonist effects include inhibition of fibroblast attachment on FN perhaps to prevent premature deposits of collagen (143). TN also displayed greater elevation in B2. Greater responses may be skewed by genotype differences. One study supports a role of TN in muscle capillarization postexercise, an important step in muscle healing. After aerobic exercise training, only individuals who were A-nucleotide carriers exhibited increase capillary to fiber ratio, while 59 no changes was reflected in T-nucleotide carriers. While the two exercise modalities differ, RET has been shown to preserve and enhance capillarization in older men (217,218). FN plays many roles in the ECM, including binding of ligand, receptors, fibrin, TGFβ-LAP complex and more. During remodeling, FN contributes to potent adhesive properties, guiding the assembly of collagens and fibrillin (219). It has been previously reported that FN immunoreactivity increases 72 h post 100 ECC contractions (84). However, in the present study, FN gene expression did not increase after B1 or B2. An increase was hypothesized considering physical trauma and lesions are marked by an appearance of FN, presumably to direct the development of granulation tissue (220). Formation of a fibrin-fibronectin matrix serves as a scaffold for deposition of new matrix via fibroblasts migration, allowing attachment prior to differentiation into myofibroblasts (221,222). FN was also reported to be unchanged after RE consisting of leg press and leg extensions (223). It was thought that the absence of upregulated TN following RE was due to the lack of or a small magnitude of damage. Despite evident damage from force loss data, gene expression did not align with previous reports of increased immunoreactivity. Mild to moderate sarcolemma injuries in humans produced by voluntary ECC contractions is unlikely to demand granulation, which may be one explanation for lack of differences in FN pre and postexercise (224,225). Taken together, the transitional matrix likely provides instructional cues or acts as quality control to ensure the organization of the matrix before laying down of collagens. However, it is uncertain the extent of damage that demands this transient matrix and the correlation between gene and protein levels. 4.1.2.3 Degradation of ECM by Matrix Metalloproteinase MMPs are the primary proteinases for the induction of collagen breakdown, likely vital for maintenance of collagen turnover. MMP2 is constitutively produced by skeletal muscle and 60 implicated during ECM remodeling for breakdown of collagens making up the basement membrane along with other matrix and non-matrix substrates (226). Animal models demonstrate that MMP2 activity coincides with myogenesis after lesion-induced damage, with maximal activation between days 3 to 10 and levels remaining upregulated up to 30 days (134,227). Moreover, a systematic review of evidence from human biopsies showed that only repeated RE stimulated MMP2 transcription (127). Elevated MMP2 at B2 corroborates with later activation, possibly for mobilizing of MuSC by degradation of the basement membrane. Elevated levels at later timepoints could also indicate higher collagen turnover, as collagen transcripts appears simultaneously without increase in total endomysial collagen (3). MMP2 mRNA levels after both ECC bouts in the control leg, however, decreased. This could be due to greater damage postponing migration and fusion of MuSc. The primary MMPs that act on collagens type I and III were not analyzed in this study, therefore it is not known whether upregulation of these proteolytic molecules symbolize this catabolic phase, though its increase likely supports building of a stronger matrix. 4.1.3 Prolonged response of the ECM to RET Effective attenuation of damage by RET suggests that mechanisms of adaptation by one maximal ECC bout are similar to that of repeated RE. Repeated RE likely resulted in temporal summation of protection to resemble one maximal ECC bout. Post-B2 force data does not discern a difference between legs. However, both legs experienced rapid recovery and minimal damage compared to previously reported data (4). Cross-education from RET to the control leg may have induced a preconditioning effect so that the magnitude of protection conferred by B1 is greater. Considering the effects of one RE bout is abolished are 14 d postexercise (40), a 21 day period between ECC bouts may have diminished the effects of the last RE session making the 61 magnitude of protection between legs similar. In other words, protection by B1 masked residues of RET. Transcriptomic studies show RET promotes similar ECM gene signatures as repeated ECC bouts (4,17,214). RET appears to accelerate COL4A1, observed by a 4-fold increase 48h post-B1. The control leg did not display any changes in COL4A1 until pre- and post-B2. COL4A1 gene and protein levels demonstrate a delayed upregulation at 27d and 22d, respectively, following a damaging bout (3,4). Furthermore, training contributes to the elevation of COL4A1 gene expression (207). Upregulation induced by 2 to 3 weeks of RET indicates ECM reconstructive activity as a common pathway between RET and RBE. Post-hoc analysis for TGFβ gene expression revealed an upregulation of TGFβ post-B1 (Cohen’s D ES = 0.67) and post-B2 (Cohen’s D ES=0.75) compared to the downregulation postexercise observed in the control leg. TGFβ plays a role in stimulating pro-fibrotic gene expression including collagens, therefore, an upregulation further supports accelerated ECM remodeling by RET. While TGFβ does not directly increase COL4A1, it may increase the number of myofibroblast by transdifferentiation from fibroblasts to stimulate continued collagen synthesis. Some studies have shown TGFβ to indirectly influence the production of COL1A1 and COL3A1 (91), however, neither gene was upregulated post-B1 in the trained leg. It was expected that RET would induce collagen gene expression by B1, however, the results show that most ECM related transcriptional changes only occurred in B2. The lack of ECM factors responding to RET may be due to contraction dependent adaptations where transcriptional changes is dependent on exercise modality. Inclusion of CON muscle actions during RET may have dampened the signatures reported by the Hydahl group that is only reflective of damaging ECC contractions (4). Training specificity is well known and has been previously observed by multiple studies (61,228,229). 62 Differing modes of contraction lead to different morphological adaptations, for instance, ECC-associated increases in fascicle length are juxtaposed to pennation angle changes after CON training (61). Early plasticity of the muscle is reflected transcriptionally by unique molecular signatures, when introduced to a new training program. ECC adaptations pertained to upregulation of ECM and immune related genes plus down regulation of mitochondrial pathways. Whereas, CON-specific down regulations were ribosome-related (230). These transcriptional responses appear to dampen after 4 weeks of training. This poses an interesting paradox, where opposing contraction or training methods may hinder the other’s adaptive pathways at least initially. Therefore, RET may have induced different gene signatures that have not yet been explored in relation to ECM remodeling. 4.2 Limitations A few limitations were present in the outlined study even though some novel and interesting findings were provided. Although muscle biopsies are often performed prior to isometric force data collection, they may have influenced the participant’s true ability to produce force at 48h post-B1 and B2. Only a few indirect measures of EIMD were examined in this study. Strength loss is decidedly representative of EIMD, but some other commonly measured markers include molecular (e.g., nuclear factor kappa B, CD68) and physical (e.g., range of motion). Even though the role of molecular factors is unclear and difficult to draw conclusions from, it would be of interest to identify markers of damage with RET. Capturing of range of motion would have strengthened how the exercises imposed affect functional performance. Direct measures to detect cellular and subcellular disturbances were also not examined, particularly by z-line streaming. 63 But, human exercised muscle does not exhibit large ultrastructural damage. Muscle damage was only quantified in 7% of fibers after 9 weeks of heavy RET (231) and 10% of fibers 24h after voluntary ECC contractions (232). When compared to rats subjected to ECC contractions, 30% of fibers were affected (233). Strength decrements represent the functional capacity of the muscle and seem to correlate with EIMD, both peaking between 24-48h after ECC exercise (234). Analysis of direct makers may supplement MVC data to provide a complete picture of both functional and structural alterations. The study was powered to detect strength loss differences and potential change in TGFβ based on data reported on the receptor, thus despite a lack of statistical significance for many comparisons of molecular markers, the current data provide important clues to transcriptional regulation of ECM-related genes. Nevertheless, utmost efforts were made to maximize the power of the study using previously reported data (4,12). Transcriptional changes were only a secondary and tertiary objective, and even though some significance and trends were identified in the current study, a larger sample size would have to be utilized to optimize detection. Additionally, changes at the transcriptional level cannot be directly extrapolated to reflect protein level changes. Post-transcriptional modification and translational regulation need to be understood and considered to fully understand these transcriptional findings. A unilateral design eliminated individual variations but may have induced cross-education where some level of protection may have crossed-over during the training period to the control leg, thus impacting susceptibility to EIMD following B1. Though 6 sessions of RET likely did not induce large strength gains, damage was still prominent and greater in the control leg after B1. Although the present study did not analyze CL-RBE, force loss data does indicate 64 some level of protection. Considering there has only been one study investigating the effects of CL-RBE on the lower leg, the present results support cross-education of RBE. 4.3 Future directions A longer duration training period, perhaps 6-12 weeks would further delineate long-term ECM responses to RET. Transcriptomic studies support an upregulation of ECM genes after long-term training (214), so it would be of interest to understand the time frame gene expression is upregulated and whether chronic training promotes the continued elevation of these genes. Furthermore, comparisons of a trained vs. untrained ECM response to acute damaging bouts, will identify whether ECM is a mechanism of RBE and whether the response to damage changes depending on the trained state of the skeletal muscle and ECM. CL-RBE is prevalent in the elbow flexors and some degree of cross-education may have been exhibited in the current study. Determining the presence of CL-RBE in the lower limbs and its mechanism would be of interest. The current consensus for CL strength gains largely stems from neural adaptations. Does the same drive CL-RBE or are there other factors? Mismatch between transcription and translation, for instance with TGFB, only allows glimpses of the interconnectedness of ECM molecules in this adaptation process. Further immunohistochemistry work, staining the transitional matrix or collagen may elucidate whether changes in gene transcription translate to the protein level. Work identifying latent TGFB after exercise could explain the relationship between latent, active and its receptor. It is assumed that the lack of change seen in the present study in active TGFB is explained by either immediate binding to the receptor or upregulation at the level of latent TGFB with dissociation from LAP 65 only when downstream pathways are required. Protein level analysis of either latent TGFB or TGFBRII may explain regulation of TGFB. In this study only MMP2 was analyzed. The MMPs implicated in degradation of interstitial collagen, I and III, is MMP14. Targeting a larger group of MMPs and their corresponding collagen gene expression will outline the time course of when degradation will occur. Presence of sex-difference indicated by animal models show that males may experience greater damage. Conversely, absence of sex difference or less damage in males is purported in humans (235). Future research on sex differences in EIMD may highlight ECM remodeling differences. 4.4 Conclusion In conclusion, 6 sessions of RE attenuated strength decrements following maximal ECC contractions. One maximal ECC bout can confer protection to a subsequent bout of the same exercise when performed 3 weeks later. ECM transcriptional changes do not appear to respond to acute bouts of exercise but display a delayed upregulation in response to both training and maximal ECC contractions. Transcription of ECM regulators appears to remain elevated with repeated RE or ECC contractions. 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Available from: http://www.ncbi.nlm.nih.gov/pubmed/12409811 85 Appendices Appendix A - Sample Muscle Function Testing Data Recording Sheet 86 Appendix B - Familiarization Session Data Recording Sheet 87 Appendix C - Sample Muscle Function Testing Data Recording Sheet 88 Appendix D - Sample Training Session Data Recording Sheets 89 Appendix E - Sample Food Log 90 Appendix F - Participant Information and Informed Consent Sheet Title of Study: Resistance Exercise Training as a Stimulus of the Repeated Bout Effect and the Contribution of the Extracellular Matrix in Young Adults (RTRBE) Principal Investigator: Assistant Professor Cameron Mitchell Ph.D., School of Kinesiology, The University of British Columbia, Phone: 604-827-2072, Email: Cameron.mitchell@ubc.ca Sponsor(s)/Funder: The University of British Columbia, School of Kinesiology Study Contact Number: 604-827-2072 ______________________________________________________________________________ General Information: You are being invited to participate in a research study investigating whether resistance training can induce the repeated bout effect. An effect whereby a single session of, usually lengthening muscle contractions, protects the muscle against muscle soreness and strength loss in future training sessions. We will also be looking at the contribution of the 3-D scaffolding that surrounds the muscle towards this effect. In order to decide whether or not you would like to be a part of this research study, you should understand what is involved and the potential risks and benefits. This form gives detailed information about the research study, which will be discussed with you. Once you understand the study, you will be asked to sign this form if you wish to participate. Feel free to discuss this consent form with your friends, family, and if needed, your health care professional. Please take your time to make your decision. At any time before or during the study, please ask the principal investigator or study coordinator to explain any words or information that may be unfamiliar to you. Once you have read this consent form and your questions have been answered, you will be asked to sign and date the last page, as well as initial each sheet of the document, if you want to participate in this study. By signing this consent form, you are stating that you agree freely and voluntarily to participate in this study, Your participation if voluntary Your participation in this study is entirely voluntary, so it is up to you to decide whether or not to take part in this study. If you do decide that you would like to participate, you are still free to withdraw at any time without giving any reason for your decision. If you do not wish to participate, you do not have to provide any reason for the decision nor will you lose the benefit of any medical care to which you are 91 entitled or presently receiving. If you are a student at the University of British Columbia (UBC) your decision to participate, not participate, or withdraw from the study will have no bearing on any course grade or future opportunities within the School of Kinesiology. Who is conducting this study? This study is being conducted by Dr. Cameron Mitchell with Donna Li working under his supervision. Dr. Mitchell does not receive any personal financial compensation for this study. This study is funded by NSERC. Why is this research being done? Postexercise symptoms including muscle soreness, pain, strength loss, and decreased range of motion all impair the ability for the muscle to function optimally. The repeated bout effect is a phenomenon that decreases both muscle soreness and pain in later exercise bouts when a prior maximal exercise bout is performed. Thus, allowing less overall damage to the muscle. Recent evidence suggests that the network surround skeletal muscle plays an important role in this effect. The main goal of this study is to determine specific changes that occur in the surrounding network in the repeated bout effect, and whether resistance training can stimulate this effect to a similar extent as a single bout of lengthening contractions. Who can participate in this study? You can participant if you can: • Able to understand and communicate in English • Aged 18 – 30 • Female or Male • Able to commit to two training sessions per week for a continuous 3-week period, as well as two maximal eccentric (lengthening) bouts and 9 days of testing in the 3 weeks following. The ability to understand and communicate in English is required because you must be able to quickly and concisely communicate with the research staff in case of an emergency or any study related discomforts and you will also be receiving exercise coaching/training advice/cues that must be fully understood and carried out in order to provide a safe and effective resistance exercise trial. You should not participate in this study if you: 1. Have diabetes, cardiovascular disease, or current pharmaceutical therapy 2. Have medical conditions impacting your ability to undertake strenuous physical activity 3. Engaged in any lower body resistance training in the previous 6 months (more than 2 h per week) 4. Engaged in any aerobic activity in the previous 6 months (more than 2 h per week) 5. BMI over 30 6. Currently smoke cigarettes daily 7. Currently taking blood-thinning medication or have a bleeding disorder 92 What will my responsibilities be if I take part in the study? If you volunteer to participate in this study, we will ask you to do the following: You will visit the laboratory of Dr. Cameron Mitchell at the Chan Gunn Pavilion on the UBC Vancouver Campus on 18 occasions. You will complete a baseline maximal voluntary contraction (MVC) strength testing for your legs, resistance exercise training sessions, undergo muscle biopsies, and perform eccentric (lengthening) contractions. A muscle biopsy is a procedure where a small incision is made in order to obtain a portion of muscle tissue from a targeted muscle. Any potential risks/discomfort are discussed on page 4 of this document. Visit 1 Familiarization/Screening – 60 min: You will be asked to attend a screening and strength testing session at the UBC Chan Gunn Pavilion. This visit will last less than one hour. When you arrive, the study will be explained to you in detail and you will have an opportunity to ask any questions you may have. We will also ask you some questions about your health and physical activity habits to make sure you qualify. The Canadian Society of Exercise Physiology Get Active Questionnaire (CSEP GAQ) will be used to assess your readiness to perform physical activity as well as identify your current level of physical activity. The CSEP GAQ Reference Document will also be made available to you if you answer ‘Yes’ to any questions on the CSEP GAQ. Then if you choose to participate, you will sign the consent form before any other study related activity. We will also go over leg press and extensions for the resistance exercise training and the eccentric contraction that will be performed. The dominant leg will be identified, and randomized to each resistance exercise training (RET) or control condition. Your RET leg will complete the 6 session of RET, while the control will not. Randomization of the leg will be assigned using and only randomization software. Visit 2,6-10 Resistance Exercise Training (RET) – 30 min: Approximately one week after your first visit, you will return to the Chan Gunn Pavilion to begin the first session of RET, this session will last roughly 30 min. After an optional warm-up on a stationary bicycle, you will be asked to perform knee extensions and leg presses on the RET leg. This will be conducted for 3 sets of 10-12 repetitions, or until volitional fatigue is reached. In the first session, you will be asked to perform a maximum voluntary contraction (MVC) on the Biodex Dynamometer. During each session, the load will be adjusted as you get stronger. There will be a 90 s rest between each set and 120 s rest between each exercise. A total of 6 sessions will be completed within 21 days (3 weeks). Thereafter you will be provided with food log sheets and be asked to record your diet 24 hours prior to each muscle biopsy, You will be ask to adhere to the same diet to the best of your abilities prior to each biopsy. Visit 3-5 Muscle Function Testing – 15 min: Approximately 24 hours after the first RET sessions, you will return to the lab to undergo muscle soreness assessment and MVC testing, in that order. You will be asked to perform a bodyweight squat movement and then draw a vertical line on a horizontal scale from no soreness to extreme soreness. After resting for 60 seconds, you will undergo MVC testing. The RET leg will be attached to isokinetic arm, while the trunk will be fixed with a strap across the chest. The isokinetic arm is the moving part of the machine, similar to a leg extension exercise machine. A warm-up set of 10 repetitions will be first performed. Thereafter, you will be asked to perform 2 MVCs, holding for 3-5s. You will be asked to use as much force as possible. Between each set, you will rest for 2 minutes. You will also be reminded to overnight fast prior to biopsy day. 93 Visit 11 Acute Eccentric Bout 1 & Muscle Biopsy 1 – 90 min: After the cessation of the RET sessions, you will be asked to return to the lab exactly 21 days after your first RET session. First, muscle soreness assessment and MVC testing will be conducted. After testing, a muscle biopsy will be collected from both thighs. You will first be locally anesthetized by a physician, then the biopsy will be taken. The area of incision will be approximately 1 cm. After collecting the biopsy of around 100mg, the physician will close the wound and apply dressing. You will then be asked to complete an eccentric (lengthening) bout on each leg for 10 sets of 10 repetitions, with 2 minutes of rest between each set. At the end of your session we will provide you with extra medical dressing in the event that the dressing applied by the physician requires changing, as well as information on how to take care of your biopsy incisions. Visit 12-14 Muscle Function Testing – 15 min: • Muscle soreness assessment • MVC testing Visit 13 Muscle Biopsy 2 – 60 min: • Muscle biopsy • Muscle soreness assessment • MVC testing Visit 15 Acute Eccentric Bout 2 & Muscle Biopsy 3 – 90 min: • Eccentric bout • Muscle biopsy • Muscle soreness assessment • MVC testing Visit 16-18 Muscle Function Testing – 15 min: • Muscle soreness assessment • MVC testing Visit 17 Muscle Biopsy 4 – 60 min: • Muscle biopsy • Muscle soreness assessment • MVC testing Overall Study Timeline Measure Visit 94 0 1 2 3-5 6-10 11 12 13 14 15 16 17 18 Initial Phone Contact x Informed Consent x CSEP-GAQ x MVC & VAS Testing x x x x x x x x x x Training Sessions x x Eccentric Bout x x Muscle Biopsy x x x x How will my muscle biopsies samples be handled? Intended use of the samples: Muscle biopsies will be used to analyze the effects of RET and eccentric contractions on the skeletal extracellular matrix adaptations. The biopsies will be analyze for both gene expression and protein content. Gene expression analysis will include genes such as FN, TN,TGF, MMP2, MMP9, Col1a1, Col1a3, Col1a5, Protein analysis will include proteins such as MMP2, MMP9 TGF. There will be no associated commercial use of the samples. Where will the samples be stored and who will be in charge of them: The samples will be stored in Dr. Mitchell’s Exercise and Metabolism Research Lab on the UBC campus. Access to the samples will be overseen by Dr. Mitchell. Measures employed to protect the privacy to the participants and the Researcher’s plan for handling foreseen results and findings: Each participant will be assigned a numerical code used to identify the participant and their samples during the study. The code rather than their name will be included on all data collection sheets. A single file will be maintained which links study codes to the participant's identity. During the data collection phase access to this file will be restricted to Dr. Mitchell and study personal who require it for scheduling purposes. After data collection, the file will be password protected and only accessible by Dr. Mitchell in the event a participant must be contacted about any foreseen findings including clinically relevant or incidental findings. Length of time the samples will be kept and disposed of: 95 It is anticipated that all muscle samples collected during the study will be consumed during analysis. Any remaining bio-specimens will be disposed of in accordance with UBC’s Environmental Services Facility regulations after a period of 10 years. During the 10 years, further analysis of the de-identified samples may be conducted. What are the potential risks and discomforts? Resistance Exercise Training Sessions: Mild soft tissue discomfort (i.e., muscle soreness) may be experienced after participating in heavy resistance exercise. If soreness does occur after exercise, it will likely resolve itself within a few days with no treatment. In the event that soreness is not resolved, please contact the associate investigator. Acute Eccentric Bouts: Muscle fatigue or discomfort may be experienced during the eccentric contractions on a dynamometer. To initiate movement, the participant must produce and maintain a certain amount of force which may be uncomfortable due to fatigue in later sets. Moderate soft tissue discomfort may be experience after the eccentric bouts, but will likely resolve itself within a few days with no treatment. Muscle Biopsies: You will not feel any sharp pain during the muscle biopsies, but may experience “a deep pressure sensation”, which may be associated with some discomfort. After the biopsy your leg may be sore for a few days. These are small risks (~1/1000) of infection, bleeding or a small area of diminished sensitivity around the biopsy. We will provide you with instructions for care of your biopsy sites to further minimize any risk of infection or bleeding. In the event that you experience a small area of reduced sensitivity, full feeling will return but this could take up to six months. When you donate your tissue for genetic testing or research, you are sharing genetic information, not only about yourself, but also about biological (blood) relatives who share your genes or DNA. The risk of your information being accidentally released in this study is estimated to be minimal risk. A recently passed Federal (Canada-wide) law now prohibits anyone such as an employer or an insurer from requiring you to disclose the results of a genetic test or to take a genetic test as condition of providing services. In addition, discrimination against individuals based upon genetic characteristics is now prohibited by the Canadian Human Rights Act What are the potential benefits? Regular exercise is beneficial for overall physical health. There are no other direct benefits. Will I be paid to participate in this study? To compensate you for your time and participation in this study you will be reimbursed up to $400 to cover any transportation expenses. Note that the reimbursement is pro-rated if you only complete the study partially and the $400 is paid in full upon completion of the study. If you choose to withdraw due to complications from the study, you will receive full compensation. What happens if I decide to withdraw my consent to participate? Your participation in this study is voluntary. You may withdraw from this study at any time without giving reason. If you choose to enter the study and then decide to withdraw at a later time, you have the right to request the withdrawal of your information and your samples collected during the study, or you 96 may choose to leave the study and allow the investigators to keep the information already collected about you until that point. If you choose to have the data collected about you destroyed, this request will be respected to the extent possible. Please note however that there may be exceptions where the data and/or samples will not be able to be withdrawn. For example, where the data and/or samples is no longer identifiable (meaning it cannot be linked in any way back to your identity), or where the data has been merged with other data. If you would like to request the withdrawal of your data and/or samples, please let an investigator know. What happens if I have a research-related injury? By signing this form, you do not give up any of your legal rights and you do not release the study doctor, participating institutions, or anyone else from their legal and professional duties. If you become ill or physically injured as a result of participation in this study, medical treatment will be provided at no additional cost to you. The cost of your medical treatment will be paid by your provincial medical plan. How will my taking part in this study be kept confidential? Your confidentiality will be respected. However, research records and health or other source records identifying you may be inspected in the presence of the Investigator and UBC Clinical Research Ethics Board (CREB) for the purpose of monitoring the research. No information or records that disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent unless required by law. You will be assigned a unique study number as a participant in this study. This number will not include any personal information that could identify you (e.g., it will not include your Personal Health Number, SIN, or your initials, etc.). This number will only be used on any research-related information collected about you during the course of this study, so that your identity will be kept confidential. Information that contains your identity will remain only with the Principal Investigator and/or designate. The list that matches your name to the unique study number that is used on your research-related information will not be removed or released without your consent unless required by law. Your rights to privacy are legally protected by federal and provincial laws that require safeguards to ensure that your privacy is respected. You also have the legal right of access to the information about you and, if need be, an opportunity to correct any errors in this information. Further details about these laws are available on request to your study doctor. To do this research, we need to collect health information that identifies you. We will collect information from activities described in the Study Procedures section of this form. If the results of this study are published or presented in public, information that identifies you will be removed. If you decide not to sign the form, you cannot be in the study. At the end of the 5-year retention period after publication, all paper records will be disposed of in confidential waste or shredded, and all electronic records will be deleted. Your de-identified research data may be published or deposited into a publicly accessible location at the time of publication. This data could include extracellular matrix protein and gene expression response, etc. At no time will identifying information, such as your name or birth date be included in such data. This means that other researchers may analyze the data for different reasons other than those described in this consent form. Once the data is made publicly available, you will not be able to withdraw your data. 97 The extent of the risk of you being identified through public data is unknown, but currently appears to be low. Who do I contact if I have any questions or concerns about my rights as a participant? If you have any concerns or complaints about your rights as a research participant and/or your experiences while participating in this study, contact the Research Participant Complaint Line in the University of British Columbia Office of Research Services by e-mail at RSIL@ors.ubc.ca or by phone at (604) 822-8598 (Toll Free: 1-877-822-8598). Please reference study identification H21-01924 when calling so the Complaint Line staff can better assist you. If I have questions about the study procedures during my participation, who should I speak to? If you have any questions or desire further information about this study before or during participation, or if you experienced any adverse effects, you can contact Donna Li at (647) 887-6183 or or Assistant Professor Cameron Mitchell at (604) 827-2072 or Final Consent I have had the study explained to me and I understand the risks and benefits involved. I may withdraw from the study at any time with no prejudice. My signature on this consent form means: ▪ I have read and understood the information in this consent form. ▪ I have had enough time to think about the information provided. ▪ I have been able to ask for advice if needed. ▪ I have been able to ask questions and have had satisfactory responses to my questions. ▪ I understand that all of the information collected will be kept confidential and that the results will only be used for scientific purposes. ▪ I understand that my participation in this study is voluntary. ▪ I understand that I am completely free at any time to refuse to participate or to withdraw from this study at any time. ▪ I understand that I am not waiving any of my legal rights as a result of signing this consent form. ▪ I understand that there is no guarantee that this study will provide any benefits to me. 98 I will receive a signed copy of this consent form for my own records. I consent to participant in this study. _______________________________ _______________ ___________ Participant Signature Date ________________________ ____________ _______________ ___________ Person Obtaining Consent Study Role Signature Date
Thesis/Dissertation
eng
Kinesiology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Mitchell, Cameron J.
Short-term resistance exercise training attenuates muscle damage and alters transcription of muscle extracellular matrix regulators in response to 300 eccentric contractions in young adults
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