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Stimuli and sensors that initiate skeletal muscle hypertrophy
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following resistance exercise
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Henning Wackerhage1)*, Brad J. Schoenfeld2), D Lee Hamilton3), Maarit Lehti4), and Juha J.
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Hulmi5)
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1) Department of Sport and Exercise Sciences, Technical University of Munich, Germany
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2) CUNY Lehman College, Bronx, NY
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3) Faculty of Health, School of Exercise and Nutrition Sciences, Deakin University, Australia
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4) LIKES Research Centre for Physical Activity and Health, Jyväskylä, Finland
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5) Neuromuscular Research Center, Biology of Physical Activity, Faculty of Sport and Health
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Sciences, University of Jyväskylä, Finland
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*Corresponding author:
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Prof. Dr. Henning Wackerhage
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Exercise Biology Group
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Department of Sport and Health Sciences | Georg-Brauchle-Ring 60/62 | 80992 Munich |
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Germany
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Email: henning.wackerhage@tum.de
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Phone: 0049 (0)89 289 24480
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Abbreviated running title: Hypertrophy stimuli & sensors
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Abstract
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One of the most striking adaptations to exercise is the skeletal muscle hypertrophy that
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occurs in response to resistance exercise. A large body of work shows that a mTORC1-
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mediated increase of muscle protein synthesis is the key, but not sole, mechanism by which
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resistance exercise causes muscle hypertrophy. Whilst much of the hypertrophy signaling
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cascade has been identified, the initiating, resistance exercise-induced and hypertrophy-
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stimulating stimuli have remained elusive. For the purpose of this review, we define an
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initiating, resistance exercise-induced and hypertrophy-stimulating signal as “hypertrophy
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stimulus”, and the sensor of such a signal as “hypertrophy sensor”. In this review we
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discuss our current knowledge of specific mechanical stimuli, damage/injury-associated and
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metabolic stress-associated triggers as potential hypertrophy stimuli. Mechanical signals are
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the prime hypertrophy stimuli candidates and a Filamin-C-BAG3-dependent regulation of
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mTORC1, Hippo and autophagy signalling is a plausible albeit still incompletely
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characterised hypertrophy sensor. Other candidate mechanosensing mechanisms are
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nuclear deformation-initiated signalling or several mechanisms related to costameres, which
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are the functional equivalents of focal adhesions in other cells. Whilst exercise-induced
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muscle damage is probably not essential for hypertrophy, it is still unclear whether and how
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such muscle damage could augment a hypertrophic response. Interventions that combine
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blood flow restriction and especially low load resistance exercise suggest that resistance
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exercise-regulated metabolites could be hypertrophy stimuli but this is based on indirect
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evidence and metabolite candidates are poorly characterised.
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Introduction
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Adequate muscle mass and strength are not only important for sporting performance but
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these attributes also are associated with good health and longevity (25, 162). For example, a
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recent analysis of the data of half a million people demonstrated that low grip strength is
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associated with a higher all-cause and disease-specific mortality as well as disease
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incidence for several major diseases (20). The key intervention to induce muscular
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hypertrophy and to make us stronger is resistance exercise in combination with nutrition. The
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current recommendation is for individuals to train with 40-80% of their 1 repetition maximum
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(1RM, i.e. the maximal weight that we can lift once) for hypertrophy, with loads >60% to
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increase maximal strength (135). Additionally, exercisers should perform multiple sets, rest
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for >2 min in-between sets and consume a diet that contains at least 1.6 g of protein per kg
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body weight per day (101).
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With respect to the muscle protein synthesis and the hypertrophic response to resistance
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exercise, the mechanistic target of rapamycin (the key mTOR containing complex is
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abbreviated as mTORC1) is a downstream hypertrophy signaling “hub” that controls protein
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synthesis (15, 94, 117). This is supported by extensive experimental evidence including
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research showing that mTORC1 blockade with rapamycin prevents or reduces the increase
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of muscle protein synthesis and/or muscle size after resistance exercise in humans (33) and
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in rodents (83) or when muscle is overloaded through synergist ablation (15, 53). Other
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signaling pathways and genes (94, 155) also regulate muscle size but their specific
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contribution to resistance exercise-induced muscle hypertrophy is incompletely understood.
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Whilst many studies have identified molecules and molecular mechanisms that regulate
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muscle mass, one key question has remained largely unanswered. It is: “What are the
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initiating hypertrophy stimuli that trigger hypertrophic signal transduction and skeletal
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muscle fiber hypertrophy in response to resistance exercise and what are their sensors?”
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Here, we define “hypertrophy stimulus” as a “first-in-line”, initiating stimulus that is of a
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sufficient magnitude and duration to trigger a skeletal muscle hypertrophic response to
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resistance exercise. Additionally, we define “hypertrophy sensor” as a sensor that senses
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hypertrophy stimuli. This definition means that hypertrophy regulators such as IGF-1 or its
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MGF splice variant are not hypertrophy stimuli because their expression change after
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resistance exercise (56) must be preceded by signaling events that alter their expression.
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Therefore, hypertrophy regulators such as IGF-1 are not “first-in-line”, initiating hypertrophy
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stimuli. So why are hypertrophy stimuli important? No matter how we vary resistance
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exercise variables such as load, repetitions or sets, it is the hypertrophy stimuli that will
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induce hypertrophic signal transduction and the resultant hypertrophy. Thus if we would
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know the actual hypertrophy stimuli then we could measure them with the goal of identifying
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interventions that maximally induce these signals.
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The aim of this review is to summarize our current understanding of candidate hypertrophy
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stimuli and sensors in three sections. First, we will discuss evidence that mechanical signals
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can act as hypertrophy stimuli after resistance exercise. In the second and third sections we
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will review evidence that exercise-induced muscle damage and metabolic signals,
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respectively, can trigger or augment a muscle hypertrophic response to resistance exercise.
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We aim to reconcile differences wherever possible and we will end with a statement of
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research directions.
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Is mechanical load a hypertrophy stimulus?
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Several reviews already discuss how mechanical stimuli could trigger a skeletal muscle
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hypertrophic response (18, 67, 127). Here we provide an update with a focus on mechanical
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stimuli of muscle hypertrophy and their sensors. Mechanical signals are arguably the most
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intuitive hypertrophy stimuli. This is based on three lines of indirect evidence. First, muscles
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atrophy when mechanical load is reduced through limb immobilization (e.g. (122), reviewed
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by (6)]. This suggests that a “normal” mechanical loading pattern is essential for baseline
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muscle mass. Second, Alfred Goldberg (51) and others have mechanically overloaded
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muscles such as the plantaris in rodents through the ablation of plantar flexor synergists, or
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cast-induced stretch. Because the overloaded muscles hypertrophied in a range of
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experimental conditions, the researchers concluded that mechanical overload is sufficient for
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skeletal muscle hypertrophy (reviewed in (57)). The issue with these studies is that the
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models used do not only alter mechanical load but additionally a host of other, potentially
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confounding variables such as metabolism, or cause damage. Third, mechanical load is also
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the key candidate hypertrophy stimulus that links human resistance exercise to skeletal
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muscle hypertrophy. This is because high forces distinguish hypertrophy-inducing resistance
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exercise from low load endurance exercise that triggers little or no hypertrophy. However, as
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we will address later, mechanical loading does not need to be excessive for muscle
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hypertrophy stimulation. Loads as low as 30% of the 1RM seem sufficient to trigger a near
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maximal hypertrophic response (2).
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The importance of mechanical load for muscle growth was demonstrated in a study where
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either young (24 ± 6 years) or older (70 ± 5 years) males completed similar work (i.e. the
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force x time-under-tension product) of leg extensor exercise at 20-90% of the 1RM. This
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study showed greater muscle protein synthesis (labelled the fractional synthetic rate, FSR) at
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higher loads peaking between 60 and 90% of the 1RM (84). A caveat to these findings is that,
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in an effort to equate workload, participants did not exercise to failure, especially when using
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lighter loads. To study the effect of different loads on muscle hypertrophy whilst training to
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failure, Lasevicius et al exercised subjects for 12 weeks using leg extension and elbow
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extension with one leg or arm at 20% 1RM and then either 40%, 60% or 80% with the
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opposite leg or arm (86). This study showed that resistance training of at least 40% of the
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1RM to failure caused a similar amount of hypertrophy as the higher load conditions. This
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finding is in line with a meta-analysis that concluded that lower load (≤60% 1RM) resistance
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training causes a similar degree of hypertrophy as higher load (>60%) resistance training
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(135). In untrained individuals even submaximal aerobic training (i.e. low mechanical load
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exercise) (77), or very low loads (16% of the 1RM) can increase muscle protein synthesis
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somewhat (4). In summary, a large amount of mainly indirect evidence suggests that
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mechanical load is a key hypertrophy stimulus associated with resistance exercise. However,
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the actual loads do not need to be excessive as loads of 30% of 1RM seem sufficient to
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trigger near maximal hypertrophic gains.
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Candidate molecular sensors that are capable of sensing mechanical load in skeletal
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muscle. Life on Earth evolved in an environment where a gravity of 9.8 m.s2 mechanically
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loaded organisms. It is therefore no wonder that living beings and their cells have not only
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evolved mechanical structures such as the muscles, the skeleton and cytoskeleton to
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withstand or overcome the pull of gravity, but also a plethora of sensors that detect
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mechanical stimuli. Such mechanosensors not only help cells to adapt to the direct force of a
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muscle fiber contraction, but also to adapt to more indirect mechanical signals such as shear
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stress, deformation, compression, and the stiffness of the extracellular matrix that surrounds
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each cell (18, 49, 145). In this section we discuss several types of candidate
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mechanosensors that allow muscle fibers to sense mechanical signals during and after
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resistance exercise, and trigger hypertrophic signaling and skeletal muscle hypertrophy.
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Mechanosensors within the skeletal muscle force transduction system. Skeletal muscle
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fibers are unique because they generate much higher forces than non-muscle cells. Single,
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skinned human type I and IIa muscle fibers have been reported to generate forces of
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532208 and 549262 µN, respectively (81), with each myosin contributing 6 pN (120).
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Non-muscle cells can also produce force through their actin-cytoskeleton but the forces are
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lower. For example, fibroblasts have been reported to produce forces of 167 µN per cell
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(79). Whilst these force values are just examples, they demonstrate that striated muscle
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fibers are unique in their high force-generating ability.
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The forces generated by the sarcomeres of a muscle fiber are transmitted to tendons and
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bones via two force-transducing systems:
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1) Forces are transmitted longitudinally from one end of a muscle fiber to the other end.
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2) Forces are additionally transmitted laterally from the sarcomere through the muscle fiber
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membrane (sarcolemma) to the extracellular matrix (141) via costameres (73) which are
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the focal adhesion equivalent in muscle fibers.
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There are several candidate mechanosensors in the skeletal muscle force transduction
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systems. For a true hypertrophy-triggering mechanosensor, a mechanism must exist by
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which force modifies the mechanosensor to trigger an early signaling response that then
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initiates hypertrophic signaling and muscle hypertrophy. Here we discuss costameres, titin
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and filamin-C-Bag3 signaling as potential mechanosensors in the force transmission systems
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of muscle fibers.
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Costamere-related mechanosensors. Historically, mechanical stimuli became a research
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focus when researchers discovered in the 1950s that cancer cells can grow on soft agar
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without anchorage whereas most non-cancer cells cannot. Researchers then discovered
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from the 1970s onwards that cells anchor the extracellular matrix through focal adhesion
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complexes that include proteins such as vinculin, talin and integrins as well as kinases
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including focal adhesion kinase or integrin-linked kinase (Ilk). Focal adhesions not only
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anchor cells on a substrate but also connect the exterior mechanically to the cytoskeleton
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and can sense and trigger adaptations to mechanical stimuli (72, 145).
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Costameres are the functional equivalent of focal adhesions in skeletal muscle. They are Z-
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disc associated structures of muscle fibers that are related to focal adhesions of other cells.
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Costameres connect the cytoskeleton to the extracellular matrix and also transmit force
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laterally from the sarcomere to the extracellular matrix. There are two costamere complexes
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which are the dystrophin-glycoprotein complex and the vinculin-talin-integrin complex.
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Costameres are clearly essential for normal muscle function as the mutation of costamere
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genes such as the dystrophin-encoding DMD gene often result in severe muscle diseases
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such as Duchenne muscular dystrophy (73). Given that these complexes function to anchor
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muscle fibers on the extracellular matrix to transmit force laterally, can they potentially
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function as sensors that sense mechanical stimuli? Is there evidence that costamere-
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associated proteins are hypertrophy sensors?
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In skeletal muscle, focal adhesion kinase (the protein is abbreviated as FAK and encoded by
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the gene PTK2) is a nonreceptor tyrosine kinase that moves to focal adhesions upon the
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adhesion of a cell to a substrate (54). In cultured C2C12 myotubes, IGF1 can increase FAK
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Tyr397 autophosphorylation and FAK is required for IGF1-induced hypertrophy and Tsc2,
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mTOR and S6K1 signalling (28). However, it is unclear whether and how FAK itself is
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activated by mechanical load during resistance exercise and whether there is a mechanical
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hypertrophy sensor that can activate FAK. At the moment there is no compelling evidence
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that FAK is directly activated by mechanical load during resistance exercise because unlike
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filamin-C or titin, FAK does not appear to have a mechano-activated protein domain.
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Moreover, 4 sets of 10 repetitions of resistance exercise did not affect activity-related FAK
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Tyr576/577 phosphorylation 6 h after exercise in fasted and fed individuals (50). However,
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phosphorylated FAK Tyr397 was increased 60-90 minutes post eccentric exercise when
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compared to concentric bout exclusively at the distal site of the vastus lateralis muscle (43).
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Generally, whilst FAK might help to regulate muscle size, there is no evidence yet that FAK
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is linked to a mechanosensor that senses mechanical load as a hypertrophy stimulus during
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resistance exercise.
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Focal adhesions are associated with phosphatidic acid-generating enzymes, such as
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phospholipases. Recently, it has been shown that mechanical stimuli in the form of
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attachment to either a soft or stiff substrate promote the conversion of phosphatidylinositol
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4,5-bisphosphate (PIP2) to phosphatidic acid. This synthesis of phosphatidic acid was
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catalyzed by phospholipase Cγ1 (PLCγ1) and activated the Hippo pathway effectors Yap
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(Yes-associated protein 1, gene Yap1) and its paralogue Taz (gene Wwtr1) (98). Yap and
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Taz are mechanosensitive (34) transcriptional co-factors that regulate gene expression
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mainly by co-activating Tead1-4 transcription factors. Yap and Taz regulate muscle
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differentiation, satellite cell function (157), are affected by many exercise-associated stimuli
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(47) and increased Yap activity in muscle fibers can cause hypertrophy (52, 159). Whilst
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these papers suggest no link to mTORC1 and even demonstrate that Yap can cause
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hypertrophy with rapamycin treatment (52), there are known links between Yap and
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mTORC1. Yap has been reported to suppress the mTORC1 inhibitor Pten (151) and to
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induce the expression of Slc7a5 and Slc3a2 that encode the Lat1 amino acid transporter (58).
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Whilst Pten expression does not decrease in the vastus lateralis 2.5 h and 5 h after human
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resistance exercise (156) and in synergist-ablated, hypertrophying plantaris muscle (21), the
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expression of the Lat1-encoding genes Slc7a5 and Slc3a2 as well as of other Yap targets
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such as Ankrd1 increases in both situations. Collectively this suggests a scenario where
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mechanical load, via an as yet unknown sensor, increases phosphatidic acid to activate Yap
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and Taz. Yap and Taz then increase the abundance of Lat1 which would sensitize the
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mechanically loaded muscle to leucine stimulation of mTORC1. However, phosphatidic acid
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not only modulates Hippo signalling but importantly for muscle, it can also activate mTORC1
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(68), which is the primary regulator of muscle protein synthesis. Indeed, hypertrophy-
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inducing eccentric contractions increased the concentration of phosphatidic acid for up to 60
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minutes in tibialis anterior muscles (109). Moreover, inhibition of phosphatidic acid synthesis
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by butanol prevents the phosphorylation of mTORC1 activity markers, suggesting that
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phosphatidic acid is a mediator of eccentric exercise-induced hypertrophic signalling (109).
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Whilst the Hornberger group first identified Z-disc-linked phospholipase D (Pld) as a
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phosphatidic acid-synthesizing enzyme (i.e. phosphatidic generating-enzymes are not only
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located in focal adhesions), they later identified a reaction catalyzed by diacylglycerol kinase
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(Dgk) as another source of phosphatidic acid in mechanically loaded muscle (164).
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Collectively, these studies suggest that mechanical stimuli can activate phospholipases to
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synthesize phosphatidic acid which in turn can activate mTORC1 and the Hippo effectors
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Yap and Taz. However, whilst these studies elucidate key signalling mechanisms in-between
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the mechanical stimulus and hypertrophy-mediating pathways, neither study identifies the
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actual mechanosensor. To identify the actual, phosphatidic acid synthesis-stimulating
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mechanosensor is a key task for future research in this area.
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Integrins are another protein group that are part of costameres. Specifically, the α7β1-integrin
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isoform (encoded by the gene Itga7) has been linked to muscle size as α7β1-integrin
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overexpressing mice have larger muscle fibers and increase muscle fiber size after eccentric
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exercise training when compared to wild-type mice. Also mTOR and its downstream target
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p70S6k are more phosphorylated at activity-related residues at rest and after eccentric
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exercise in α7β1-integrin overexpressing mice (167), suggesting that α7β1-integrin might help
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to activate mTORC1 signaling in response to exercise. However, it is unknown whether and
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how α7β1-integrin is activated by a mechanical hypertrophy stimulus during resistance
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exercise, and how α7β1-integrin then activates mTORC1 and other signaling proteins that
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cause the muscle fiber to hypertrophy.
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Costamere-based mechanosensors may also sense two additional types of mechanical
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stimuli that have been discussed as hypertrophic triggers in the more applied literature. The
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first stimulus is muscle fiber swelling, which is known as the “pump” by exercisers. The
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second potential mechanical stimulus is a change in the stiffness of the extracellular matrix
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as a result of resistance exercise. We will briefly discuss these two potential mechanical
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stimuli here. Resistance exercise results in a temporary perception frequently described as a
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“pump,” which is interpreted as muscle fiber swelling (134). Moreover, exercise-induced
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muscle damage (EIMD) can also lead to muscle swelling (116), although the associated
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edema from EIMD can last far longer than the “pump”. Whilst little definite evidence exists for
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actual muscle fiber swelling (i.e. a swelling of the muscle fiber and not of the interstitium)
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after resistance exercise, at least the whole muscle can swell as a result of single-bout of
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resistance exercise (39). In primary rat myotubes, swelling brought about by culture in a
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hypoosmotic culture medium increases glutamine uptake by 71% when compared to isotonic
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culture medium. This is dependent on integrins and the cytoskeleton, as integrin or
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cytoskeleton inhibitors prevent this effect (90). Together these data suggest that
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differentiated muscle can respond to cell swelling with increased glutamine uptake and that
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this depends on integrin or cytoskeletal loading. Such glutamine intake is potentially
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important, as it is a requirement for the uptake of protein synthesis-stimulating essential
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amino acids such as leucine (105). However, it is unknown whether the duration and extent
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of swelling is sufficient to load the cytoskeleton and that such cytoskeletal loading does not
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only induce glutamine uptake but also protein synthesis for up to three days post resistance
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exercise (99). Muscle swelling also occurs up to several days after exercise-induced muscle
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damage (166) at a time when muscle protein synthesis should have returned to baseline (99).
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Given that costameres are the sites where the cytoskeleton connects to the extracellular
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matrix and where mechanical signals can be sensed, it seems likely that any fiber swelling
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exerts a strain on costameres which then could trigger the hypertrophy response.
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Titin (gene: Ttn). Titin is a giant protein that is essential for muscle function and human
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health as mutations in the titin-encoding Ttn gene cause various human genetic diseases
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including myopathies (130). Titin spans half a sarcomere, from the Z-disc at the end of a
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sarcomere to the M-line in the middle (82). The I-band-spanning portion of titin is elastic and
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contributes to the elasticity of a passively stretched muscle. The M-line portion of titin
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contains a stretch-activated kinase. The kinase within the titin protein is activated when a
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stretch pulls several amino acids out of a so-called ATP-binding pocket, allowing ATP to bind.
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ATP binding then causes titin to tyrosine-phosphorylate itself, which in turn activates the
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kinase within the titin protein (124). Because of its stretch-activated kinase and association
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with numerous other proteins, titin has been proposed to be an exercise-related
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mechanosensor (82). Using our terminology, mechanical load would be the hypertrophy
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stimulus and titin the hypertrophy sensor.
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So what is the evidence for titin being a mechanical hypertrophy sensor? There are two
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points to consider. First, titin lies parallel to the force-generating actin-myosin proteins. This
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means if myosin and actin generate force and shorten a muscle fiber then titin will go slack.
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Consequently, the forces within a titin molecule should actually decrease rather than
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increase during a concentric contraction. Thus, titin cannot be a true force sensor in this
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situation. However, at longer muscle lengths titin forces increase and titin unfolds (62) and so
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this might activate titin kinase and trigger downstream signaling events. Related to this,
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resistance training at longer muscle lengths may cause a greater hypertrophy when
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compared to resistance training with shorter muscle length (96, 107).
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Second, whilst many signaling interactions have been reported for titin (82) there is not yet a
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convincing link between titin and mTORC1 signaling, which is the primary mediator of the
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muscle hypertrophy response to resistance exercise (see above). However, some titin
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signaling interactions are related to protein turnover through Murf1/2-proteasome and
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autophagy signalling and thus could regulate some aspects of muscle hypertrophy (82). In
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conclusion, whilst titin is a mechanosensitive skeletal muscle protein with a kinase domain it
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seems unlikely that it is the major mechanical hypertrophy sensor during standard resistance
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exercise, except perhaps at long muscle lengths.
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Filamin-C Bag3 (genes: Flnc and Bag3). Bag3 and filamin-C are proteins important for
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muscle function as mutations of these proteins cause severe myofibrillar myopathies (137).
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Filamin-C and Bag3 localize to the Z-disc in human muscle (153). Here, we discuss evidence
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that filamin-C and Bag3 form mechanosensor complex that is capable of activating mTORC1,
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the Hippo effector YAP1 and autophagy (see Figure 1). Filamins are mechanosensitive,
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actin-crosslinking molecules. In skeletal muscle, filamin-C is the major filamin located at the
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Z-disc (137). Filamins form V-shaped homodimers and forces of 5-20 pN deform the so-
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called domain pair 20-21 (128). One myosin head generates a force of 6 pN (120) and thus
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actin-linked filamins should deform if sufficient myosin heads pull on the actin to which the
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filamins are attached.
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In addition, filamins bind multiple proteins including the androgen receptor (112), which
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influences muscle size (71), and the Z-disc linked protein Bag3 (153) which has been
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proposed to sense the mechanical loading of filamin (152). However, how a mechanically
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loaded filamin dimer activates Bag3 is still unclear. Assuming that mechanically loaded
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filamin-C can activate Bag3, how could Bag3 trigger a hypertrophic signaling response?
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BAG3 connects through its WW domain (WW stands for the two tryptophanes that are
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separated by 20 amino acids (142)) to proline-rich motifs (e.g. PPxY motifs) of other
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proteins to potentially regulate three muscle hypertrophy-associated functions:
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1) mTORC1 signaling. The WW domain of Bag3 binds the proline-rich motif of the
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mTORC1 inhibitor tuberous sclerosis 1 (TSC1). So the hypertrophy-inducing mechanism
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might be that Bag3 sequesters TSC1 away from mTORC1, resulting in mTORC1
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activation and increased protein synthesis in response to mechanical loading (75).
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2) Hippo signaling. Bag3 sequesters through its WW domain proteins such as LATS1 and
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AMOTL1 which normally inhibit the Hippo effector YAP (152). As a consequence, YAP
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will be more active in mechanically loaded muscle, which is relevant for muscle size
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because increased YAP activity in muscle fibers can elicit muscle fiber hypertrophy (52,
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160).
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3) Autophagy. Bag3 binds synaptopodin-2 (Synpo2) to regulate chaperone-assisted
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selective autophagy (CASA) of damaged Z-disc proteins (7, 154). This might contribute to
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the increased autophagy (61) and rate of protein breakdown seen after resistance
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exercise (149), a process that may be important in full and functional muscle hypertrophy.
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Phosphoproteomic studies have shown that both Filamin-C and Bag3 change their
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phosphorylation after high intensity exercise in human muscle (65) and after maximal
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intensity stimulation of mouse skeletal muscle (121). This suggests that Filamin-C and Bag3
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are additionally targeted by currently unknown kinases and phosphatases that might further
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help to regulate Bag3 activity in a contracting skeletal muscle.
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The aforementioned Bag3-focussed hypertrophy stimulus-sensing mechanisms are
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illustrated in Figure 1.
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Please insert Figure 1 here.
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In summary, a filamin-Bag3-mTORC1/YAP/autophagy signaling cascade is a plausible but
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far from completely characterised mechanism by which mechanical loading during resistance
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exercise could stimulate hypertrophic signaling and skeletal muscle hypertrophy. However,
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whilst physiological, mechanical forces will probably deform a filamin homodimer, it is unclear
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how this then activates Bag3 and other hypertrophic signaling. Also the kinases and
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phosphatases that phosphorylate and dephosphorylate Filamin-C and Bag3 during exercise
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are currently unknown and it remains unclear as to how such phosphorylation affects
374
Filamin-C and Bag3 function and muscle size. This is clearly another important area for
375
future research.
376
377
Nuclear deformation and signal transduction. In muscle fibers, myonuclei are surrounded
378
by thick tubulin filaments (17) and by intermediate desmin filaments (126). These filaments
379
not only anchor myonuclei to the cytoskeleton but also expose them to forces when the
380
cytoskeleton is loaded (8) either by a passive stretch, by an active contraction or by muscle
381
fiber swelling. For example, when muscle fibers are passively stretched, myonuclei
382
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12
subsequently deform (113). Intriguingly, such nuclear deformation has recently been
383
identified as a mechanism by which mechanical load causes the Hippo effector Yap and
384
potentially other proteins to translocate from the cytosol to the nucleus (37). Given that
385
increased YAP activity can induce muscle fiber hypertrophy (52, 160), this might be a
386
mechanism by which mechanical loading could contribute to skeletal muscle growth.
387
Together the above filamin-Bag3-YAP and nuclear deforming-YAP-mTORC1 signaling
388
cascades are plausible mechanisms by which a mechanical hypertrophy stimulus could be
389
sensed and trigger hypertrophy signaling. However, there are two caveats to this hypothesis.
390
First, YAP-induced muscular hypertrophy is comparatively small and seems to be
391
independent of mTORC1 as it can occur when mTORC1 is blocked with rapamycin (52).
392
Second, myonuclear deformation has so far only been demonstrated for passive stretch
393
(113), and not for an active, shortening contraction. Nevertheless, proteins that sense nuclei
394
deformation to activate Hippo signaling should be characterized in the future.
395
396
Another type of mechanosensor are stretch-activated ion channels encoded by the genes
397
PIEZO1 and PIEZO2. Spangenburg and McBride demonstrated that broad, non-specific
398
inhibition of stretch-activated ion channels in rats in vivo with streptomycin or gadolinium
399
could attenuate the load-induced activation of mTORC1 (140). However, the expression of
400
PIEZO1/2 stretch-activated ion channels is among the lowest in human skeletal muscle when
401
compared to other tissues (www.gtexportal.org, see (97)) and so the effect might not depend
402
on the inhibition of PIEZO1/2 channels in skeletal muscle. For that reason we do not discuss
403
stretch-activated ion channels further.
404
405
In summary, there are several plausible but far from completely characterised mechanisms
406
by which mechanical hypertrophy stimuli could activate mechanical hypertrophy sensors
407
after a bout of resistance exercise. It may well be that there are several mechanical
408
hypertrophy stimuli (e.g. the contraction force, loading of the cytoskeleton and the
409
mechanical properties of the extracellular matrix) and sensors as has previously been
410
proposed by the Hornberger group (45). To date no mechanism is fully characterised as
411
either the mechanosensing mechanism or the link to mTORC1 or other hypertrophy-
412
regulating signaling proteins is incompletely described in skeletal muscle. Research into such
413
mechanisms is further hampered by the fact that the knockout of putative mechanosensors
414
such as Bag3 not only abolishes a potential hypertrophy response to resistance exercise, but
415
often leads to severe myopathies and dystrophies. This means that researchers can in many
416
cases not use global knock out animal models to test whether these proteins are essential for
417
the hypertrophy response to exercise.
418
419
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13
Is exercise-induced muscle damage a hypertrophy stimulus?
420
The possible role of exercise-induced muscle damage (EIMD) as a hypertrophy stimulus has
421
been discussed and studied since it was proposed in the 1990’s (29, 38, 132). EIMD is
422
damage that is triggered when individuals engage in new types of exercise, especially
423
lengthening or eccentric contractions conducted with a large range of motion (115, 132).
424
However, there is usually little EIMD when already resistance-trained individuals lift weights
425
due to the “repeated bout effect”. EIMD is associated with microscopic, structural changes
426
such as Z-line streaming in skeletal muscle myofibrils. This is then usually followed by a local
427
inflammatory response, disturbed Ca2+ regulation, activation of protein breakdown and
428
increased levels of proteins such as creatine kinase in the blood that escape or are secreted
429
from damaged muscle fibers (23, 76, 115). In their review, Hyldahl and Hubal (2014) propose
430
a continuum of skeletal muscle fiber damage after eccentric exercise that spans possible
431
adaptive cell signaling responses to pervasive membrane damage and tissue necrosis as the
432
most severe form of EIMD (69).
433
434
Evidence from human studies for EIMD as a hypertrophy stimulus. Although some
435
authors have endeavored to test whether EIMD contributes to muscle hypertrophy, the
436
results of these interventions are difficult to interpret. This is because the manipulation of
437
resistance training parameters to alter EIMD can also directly affect muscle mass, not just
438
EIMD. Therefore it is difficult to separate the effect of EIMD on muscle hypertrophy from the
439
effect of the confounding factors. For instance, training at long muscle lengths (i.e. the
440
stretched position) is not only associated with a greater magnitude of EIMD (11, 115) but
441
also possibly with increased muscle hypertrophy when compared to exercising with short
442
muscle lengths, at least in some muscles (14, 107) . However, this may not be due to EIMD
443
but due to the larger force production at longer fascicle lengths (40). Similarly, eccentric
444
muscle actions not only increase EIMD but also cause a slightly larger hypertrophic response
445
than concentric muscle action (32, 136). Again, it is unclear whether this is due to a higher
446
dose of an EIMD-associated hypertrophy stimulus after eccentric exercise (100) or simply
447
due to a confounding factor such as increased training load (36, 100). Collectively some
448
studies suggest a connection between EIMD and muscle hypertrophy but this could be due
449
to confounding factors.
450
451
In contrast, other studies show that the extent of muscle damage does not correlate with
452
muscle protein synthesis (48) or the magnitude of hypertrophy. Severe EIMD does not give
453
any further benefit on hypertrophy but rather attenuates it (42). Flann et al compared muscle
454
hypertrophy of naïve and pre-trained group with the same cumulative workload. The pre-
455
trained group did not experience EIMD as judged by plasma creatine kinase levels and
456
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14
muscle soreness but increased muscle strength and volume at the same magnitude as the
457
naïve group suggesting that EIMD is not essential for hypertrophy (41). However, in an effort
458
to reduce EIMD, the pre-trained group performed an additional three weeks of resistance
459
training, which may have confounded results.
460
461
A final argument against EIMD as a hypertrophy factor is that EIMD also occurs after
462
exercise that does not typically induce hypertrophy. For example EIMD occurs after
463
endurance exercise with an eccentric component such as marathon running (63) but damage
464
in these situations alone does not seem to cause muscle hypertrophy. If anything, marathon
465
running decreases muscle fiber size (150). However, these data are again difficult to interpret
466
as endurance athletes may have a low trainability for muscle hypertrophy and their long-
467
duration exercise, combined with low energy availability, may excessively activate AMPK and
468
thereby inhibit mTORC1 (70) for long periods. In summary, it is difficult to conclude based on
469
indirect human studies whether and how EIMD contributes to muscle hypertrophy. The key
470
reason for this is that it is virtually impossible to separate direct EIMD stimuli from
471
confounding stimuli that co-occur with EIMD.
472
473
Muscle damage or increased regeneration alone may induce muscle hypertrophy. The
474
regeneration of skeletal muscle after injury is a (re-) growth process but can injury per se
475
promote muscle fiber hypertrophy? In mice, severe injury of mouse tibialis anterior muscles
476
e.g. through cardiotoxin injection results in larger, but fewer muscle fibers when compared to
477
uninjured fibers (59), suggesting that injury alone is sufficient to trigger the hypertrophy of
478
some muscle fibers. A caveat is that we do not know whether the larger fibers are
479
hypertrophied, regenerated fibers or whether these are new but muscle fibers that are larger
480
than the previous muscle fibers. There is some evidence that injured muscle fibers and their
481
satellite cells can contribute to hypertrophy as transplanting muscle fiber-associated satellite
482
cells into a recipient muscle whilst inducing injury results in a near-lifelong muscle
483
hypertrophy (55). Together, these data suggest that injury alone and the combination of
484
injury and more satellite cells can lead to the development of larger muscle fibers or induce
485
muscle fiber hypertrophy.
486
487
Satellite cells, EIMD and muscle hypertrophy. Satellite cells are the resident stem cells of
488
skeletal muscle (131) and add nuclei to adult muscle fibers after resistance training (24).
489
Although non-damaging exercise can activate satellite cells to proliferate (27), satellite cell
490
activation and proliferation is larger after exercise that induces EIMD (26). In humans,
491
individuals that responded with greater hypertrophy to a resistance training programme also
492
added more myonuclei, presumably derived mainly from satellite cells, than individuals that
493
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15
responded less with less hypertrophy to the same training programme (118). This suggests
494
that the ability of satellite cells to proliferate and to add new myonuclei to muscle fibers might
495
limit muscle hypertrophy. However, satellite cells may expand especially in response to
496
EIMD to have a role in muscle repair and less so to increase myonuclei when muscle
497
actually hypertrophies, at least in the early stages of muscle growth (30).
498
499
The causal role of satellite cells on muscle hypertrophy has been investigated in mice. It
500
seems that the initial hypertrophy in response to mechanical overload can occur in wildtype
501
and satellite cell-depleted muscles (95, 103). However, the initial hypertrophy cannot be
502
maintained for months when satellite cells are depleted (46). Other research suggests that
503
satellite cells are also required for the initial hypertrophy at the muscle fiber level (35).
504
Collectively these studies show that satellite cells are essential for full skeletal muscle
505
hypertrophy over time and that satellite cell numbers and myonuclei increase after resistance
506
training. It is not, however known whether EIMD is essential in the long run to induce satellite
507
cells to proliferate and in turn trigger a muscle hypertrophic response to resistance training.
508
509
However, our main question in the present review is not whether satellite cells are essential
510
for hypertrophy but: How do hypertrophy stimuli activate satellite cells in the first step and
511
how do activated satellite cells cause muscle fiber hypertrophy in a second step? According
512
to our definition, the EIMD-related hypertrophy stimulus would be the repeated mechanical
513
load that causes muscle damage in a susceptible muscle. A damage-associated stimulus
514
would then activate satellite cells in the first step. There are too many possible stimuli
515
activating satellite cells to be effectively covered in this review. Currently the strongest
516
candidate pathway to activate quiescent satellite cells to proliferate following injury as well as
517
after exercise or mechanical stretching is the nitric oxide-metalloproteinase-hepatocyte
518
growth factor (HGF) pathway (147). Whether these stimuli activate satellite cells in a context
519
of resistance exercise bout especially after EIMD is unknown.
520
521
Other potential EIMD-associated hypertrophy stimuli and their sensors. EIMD is
522
associated with potential hypertrophy stimuli such as amino acids that result from protein
523
breakdown or factors linked to the immune and inflammatory response to EIMD and to
524
satellite cells. As a consequence of EIMD, inflammatory cells enter muscles and produce
525
substances including myokines such as IL-6 that have been reported to be able to both
526
increase (138) or decrease muscle size (10) in different contexts. The inflammatory response
527
to EIMD is thought to also induce cyclooxygenase production, which may aid hypertrophy as
528
non-steroidal anti-inflammatory drugs (NSAID, which target cyclooxygenase) blunt
529
hypertrophy following regimented resistance training (88). There is also evidence that
530
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16
reactive oxygen species (ROS) promote hypertrophy, as antioxidant supplementation can
531
blunt hypertrophic signaling (114) and reduce the magnitude of exercise-induced muscle
532
hypertrophy (12). However, even if IL-6 and ROS can influence muscle size, they are clearly
533
middlemen in the hypertrophic process, as there must be upstream hypertrophy stimuli and
534
sensors that increase their concentration in response to resistance exercise. Moreover, ROS
535
are not only induced by EIMD but also by endurance running (125), which does not typically
536
cause hypertrophy. In summary, the evidence suggesting that EIMD is associated with
537
hypertrophy is mostly indirect, some is contradictory, and putative mechanisms and sensors
538
are incompletely characterised.
539
540
Is metabolic stress a hypertrophy stimulus?
541
We have already mentioned that mechanical forces are probably the most important
542
hypertrophy stimuli. When mechanical forces are absent or reduced, other signals typically
543
only have small effects on muscle size. For example, when post-operative brace-immobilized
544
knee surgery patients intermittently occluded their thighs, their muscles atrophied by 7%
545
within 14 days which was significantly less than the 15% atrophy seen in the no occlusion
546
controls (146). This experiment suggests that potential occlusion-related hypertrophic stimuli
547
cannot compensate for the loss of mechanical loading but that they can limit atrophy.
548
However, when combining vascular occlusion with dynamic muscular contractions, marked
549
hypertrophy invariably occurs, even when employing relatively light loads or no external
550
loads at all (1, 89). In these training regimes, the vascular occlusion increases metabolic
551
stress as judged by the drop in phosphocreatine (PCr) and pH (143). Similarly, muscles
552
hypertrophy more if resistance training with relatively heavy load is conducted under
553
intermittent hypoxia versus normoxia (85, 93, 106). The fact that blood flow restriction and
554
hypoxia affect metabolism has led some researchers to suggest that metabolic stress-
555
associated signals such as metabolites (i.e. molecules involved in metabolism that are
556
typically below 1500 Da) may have an anabolic effect and contribute to muscle hypertrophy
557
(133). An alternative proposal is that “metabolites simply augment muscle activation and
558
cause the mechanotransduction cascade in a larger proportion of muscle fibers” (31). This is
559
another way of saying that some fibers fatigue during contraction, which is linked to changes
560
in metabolite concentrations such as a drop of phosphocreatine or increase of lactate. As a
561
consequence, additional fibers need to be recruited to sustain force output and these
562
additional fibers are then additionally exposed to hypertrophy stimuli. However, recent work
563
found that the addition of blood flow restriction training to a traditional resistance training
564
program preferentially enhanced type 1 fiber cross sectional area in a cohort of elite
565
powerlifters (13). This seemingly refutes the hypothesis that the hypertrophic effects of blood
566
flow restriction training are simply a function of increased high-threshold motor unit
567
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17
recruitment, and raise the possibility that the associated metabolite accumulation may induce
568
anabolism via other mechanisms. Henceforth, we discuss the potential role of metabolites as
569
hypertrophy stimuli.
570
571
Metabolic stress. Metabolic stress can be defined as the changes in energy metabolism
572
and metabolites that occur during non-steady state muscle contractions. Non-steady state
573
contractions are contractions where not all of the hydrolyzed ATP can be resynthesized by
574
oxidative phosphorylation alone. As a consequence, the concentration of PCr will
575
continuously decline as PCr resynthesizes ADP to ATP via the Lohmann reaction
576
(PCr+ADPATP+creatine). Moreover, the lactate concentration will rise and the pH will drop
577
as ATP is additionally resynthesized through glycolysis. Thus, a low PCr concentration, a
578
high lactate concentration and a low pH are biomarkers for metabolic stress. In relation to
579
these metabolites, blood flow restriction will not change the rate of ATP hydrolysis but it will
580
reduce oxygen delivery and oxidative ATP resynthesis, which requires greater PCr
581
breakdown and a higher rate of glycolysis in active muscle fibers (143, 144).
582
583
Metabolic stress during resistance exercise versus other types of exercise. The higher
584
the exercise load, the more ATP will be hydrolyzed per second and the faster PCr, lactate
585
and the pH will change. Thus, during high intensity resistance exercise, the PCr
586
concentration and the pH will drop more per second than during low load resistance exercise
587
(143, 144, 158). However, as metabolic stress either causes fatigue or is associated with it
588
(5), metabolic stress will be higher at the end of a longer duration set with low loads because
589
we can lift a lower load with a more fatigued muscle than during a shorter duration set with
590
high loads as we can only lift a high load if fatigue and metabolic stress are low.
591
592
The logic that a set with lower loads to exhaustion will cause more metabolic stress than a
593
set with heavy loads is supported by experimental data. In a biopsy study, Tesch et al
594
measured intramuscular PCr and other metabolites in the vastus lateralis before and after
595
several sets of 10 repetition leg muscle contractions to failure in trained bodybuilders.
596
Intramuscular PCr decreased from 21.33.7 mmol/kg pre exercise to 10.92.5 mmol/kg (51%
597
of pre-exercise) after the last set of exercise (148), suggesting moderate metabolic stress. In
598
contrast, during intermittent resistance exercise with 25% of the 1RM which is suboptimal for
599
hypertrophy, PCr decreased to 17±12% of the pre-exercise concentration in adult women
600
and 18±16 % of the pre-exercise concentration in adult men, respectively (74), suggesting
601
high metabolic stress. Similarly, PCr decreased from 15.81.7 mmol/kg to 1.70.4 (11% of
602
pre-exercise) after a 400 m run (64). Collectively this shows that metabolic stress is typically
603
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18
greater during non-steady state exercise with intensities that are sub-optimal for hypertrophy
604
(86) than during “classic” 10 repetition resistance training in trained individuals.
605
606
Metabolites that have anabolic signaling properties. Metabolic stress is a vague concept
607
given that 2700 metabolic enzymes catalyze 900 metabolic reactions (129) and that 4000
608
metabolites can be detected in human serum alone (123). So given the plethora of
609
metabolites, are there any metabolites or other metabolic stress-related factors that can act
610
as hypertrophy stimuli? Are there any metabolites that can be considered to be hypertrophy
611
stimuli according to our definition?
612
613
Lactate is a key biomarker for metabolic stress, used as a biomarker for performance, and
614
one of the most studied exercise metabolites. There is some evidence that lactate may affect
615
muscle differentiation and have some anabolic effects (104). In the most extensive study to
616
date, lactate affected the expression of regulators of muscle differentiation in vitro. Also the
617
authors found that a combination of a 30 min low-intensity running training program together
618
with a dose of lactate and caffeine increased muscle mass and hypertrophic signaling in rats
619
(111). It is not possible to conclude, however, how much of the hypertrophy was due to
620
lactate. Other studies suggest that skeletal muscle may sense changes in extracellular
621
lactate. For instance work form George Brooks’ lab demonstrated that when 20 mM lactate
622
caused L6 rat myotubes to express lactate-related genes (60), but this did not show that
623
lactate is a hypertrophy-stimulus. More recently, Ohno et al found that 20 mM lactate was
624
able to induce anabolic signaling and hypertrophy in C2C12 cells, possibly in a GPR81
625
dependent manner (110). This suggests that extracellular lactate can initiate signaling events
626
through membrane-bound receptors in skeletal muscle. Whilst these data indicate that
627
lactate may be a modifier of muscle signaling and hypertrophy, lactate concentrations are
628
typically highest during exercise that is suboptimal for hypertrophy such as a 400 m run.
629
630
Another anabolism-related energy metabolite is α-ketoglutarate which is not only a citrate
631
cycle metabolite but also a nitrogen scavenger (163). Long term supplementation for 9
632
weeks of the drinking water with 2% α-ketoglutarate resulted in significant gastrocnemius
633
skeletal muscle hypertrophy and increased markers of mTORC1 activity (19), suggesting that
634
α-ketoglutarate could stimulate muscle hypertrophy. In contrast, however, L-arginine α-
635
ketoglutarate supplementation did not increase strength measures such as the 1 RM after a
636
resistance training program in humans when compared to placebo control (161).
637
638
Other anabolic metabolites are phosphatidic acid and lysophosphatidic acid, which can
639
activate mTORC1 (68, 139) and Hippo (165) signaling, respectively. We have already
640
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19
discussed in the mechanotransduction section that hypertrophy-inducing eccentric
641
contractions increase the phosphatidic acid concentrations in tibialis anterior muscles (109).
642
643
Another potential source of hypertrophy-inducing metabolites is from muscle protein
644
breakdown. The activation of skeletal muscle protein synthesis by resistance exercise seems
645
to be correlated to the activation of skeletal muscle protein breakdown (119). Cell based
646
experiments demonstrate that simply increasing the intracellular concentration of key amino
647
acids like leucine by as little as 7% is sufficient for half maximal activation of mTORC1 (22).
648
Additionally, a single bout of resistance exercise in rodents causes an approximate 25%
649
increase in the intramuscular leucine concentration (91). It is theorized that this increase in
650
intracellular leucine, possibly from protein breakdown, is sensed by the amino acid sensor
651
mVPS34 leading to mTORC1 activation (91). However, feeding 40 g of protein can almost
652
triple the intracellular leucine content in human skeletal muscle (92) and it seems unlikely
653
that the small transient changes in intramuscular leucine as a result of resistance exercise
654
make a major contribution to the hypertrophy response to resistance exercise
655
656
In addition to metabolites, metabolic enzymes might also be involved in hypertrophy
657
signaling, too. Researchers found in HEK293 cells and fibroblasts that the glycolytic enzyme
658
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibit mTORC1
659
signaling. However, when glycolytic flux is high as would be at the end of a set of resistance
660
exercise then GAPDH no longer inhibited mTORC1 and cells grow (87). In this scenario, the
661
signals that activate glycolytic enzymes such as phosphorylase and phosphofructokinase
662
would be the hypertrophy stimuli and the enzymes would be their sensors. This shows a
663
plausible mechanism by which a signal related to glycolytic flux might act as a hypertrophy
664
stimulus capable of activating mTORC1 and skeletal muscle hypertrophy. These and further
665
links between metabolism, muscle mass and regeneration have recently been reviewed (78).
666
667
Studies that do not support energy stress being a hypertrophy stimulus. During
668
evolution, mechanisms evolved that reduce protein synthesis and cell growth when there is
669
metabolic stress. For example, when the metabolic stress-mimicking AMPK activator AICAR
670
was given to rats then muscle protein synthesis reduced significantly to 55% of the protein
671
synthesis measured in control rats (16). Soon after the Guan group demonstrated that the
672
metabolic stress sensor AMPK inhibited mTORC1 via TSC2 (70). Consistent with this, the
673
synergist-ablated plantaris hypertrophied more in AMPKα1 knockout than wildtype control
674
mice suggesting that energy-stress activation of AMPK can blunt hypertrophy at least in
675
some hypertrophy models (102). However, whilst prolonged metabolic stress might work
676
through such mechanisms to explain reduced muscle hypertrophy during concurrent
677
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20
endurance and resistance training (9), it is unclear whether these studies explain what
678
happens during short term metabolic stress during acute resistance exercise, which might
679
exert its effect via different metabolites and signaling molecules.
680
681
Overall summary, conclusions and directions for future research
682
Whilst there is a large amount of mainly indirect evidence about hypertrophy stimuli and their
683
sensors, this evidence is often difficult to interpret and as a consequence many questions
684
remain. Mechanical stimuli stand out as the most likely and most potent hypertrophy stimuli
685
and several potential mechanosensing mechanisms have been partially characterised. To us,
686
a key question is whether muscle fibers, which are the cells that produce the highest forces,
687
have their own specific mechanosensing system in addition to the generic focal adhesions
688
(i.e. costameres in muscle) that sense the mechanical environment of most cells. The Z-disc
689
is a prime striated muscle-specific candidate site for muscle-specific force sensing. Z-discs
690
are not only directly exposed to the forces generated by sarcomeres but Z-discs additionally
691
transmit these forces longitudinally and laterally via costameres (44). Moreover, the Z-disc
692
becomes a signalling hub when muscles contract with high intensity and generate large
693
forces. This is supported by the results of a recent phosphoproteomic study which reported
694
that the majority of Z-disc proteins robustly alter their phosphorylation in response to maximal
695
intensity contractions of mouse muscles. In particular, the Z-disc localized kinases obscurin
696
and Speg change their phosphorylation and the Z-disc localized Filamin-Bag3 complex
697
proteins are also phosphorylated (121, 153).Thus future studies should seek to answer the
698
question “Is it mainly the Z-disc or the costameres where mechanical hypertrophy stimuli are
699
sensed and transduced after resistance exercise?”
700
701
Data suggesting and supporting EIMD or metabolic stress-related hypertrophy stimuli are
702
mostly indirect and the related molecular mechanisms are poorly understood. Moreover,
703
growth can occur in the relative absence of either of these putative signals, lending further
704
support for the hypothesis that mechanical stimuli are the primary hypertrophy stimuli. That
705
said, research indicates that both EIMD and metabolic stress regulate multiple factors
706
involved in the hypertrophic process, and a sound rationale exists whereby their resistance
707
training-induced manifestation may contribute to hypertrophic adaptations. If so, it remains to
708
be determined whether these factors are additive to mechanically-derived signaling or
709
perhaps redundant provided a given level of mechanical force is achieved. Moreover, if these
710
signals are indeed additive, it remains to be determined whether an upper threshold exists
711
beyond which no further growth-related benefits are realized. In particular, any hypertrophic
712
effects of EIMD would almost certainly follow a hermetic curve, with benefits seen only up to
713
a given point and they ultimately inhibit hypertrophy when EIMD is excessive. To this point, a
714
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21
high degree of EIMD impairs a muscle’s force-producing capacity, which in turn interferes
715
with an individual's ability to train as well as negatively impacting recovery (80, 108). Thus,
716
there may be a “sweet spot” whereby a combination of mechanical, metabolic, and damage-
717
related signals interact synergistically to promote a maximal hypertrophic response.
718
719
Finally, how to proceed towards the long-term goal of identifying all major hypertrophy stimuli
720
and their sensors? It is clear that the leading researchers must move beyond indirect
721
association studies as there are just too many confounding variables to draw valid
722
conclusions. Force, metabolism and EIMD are all linked and it seems impossible to vary only
723
one of these variables during resistance exercise without varying the others and so such
724
studies are never fully conclusive. One key experiment is to assess whether a putative
725
hypertrophy sensor is essential for the muscle hypertrophy adaptation to resistance exercise.
726
To test for this, the gene that encodes the sensor needs to be knocked out or inhibited
727
pharmacologically to evaluate whether this prevents adaptation to exercise. However, the
728
problem with this approach is that putative hypertrophy sensors such as Bag3 are essential
729
for normal muscle function (66). Hence, their global knock out typically causes a myopathy or
730
dystrophy, which limits the usefulness of such models for studying their role in hypertrophy
731
signaling. Here, more sophisticated transgenic animal models are needed. Strategies could
732
involve targeting the transgenesis to skeletal muscle only, making it inducible and modulating
733
solely those sites of a protein that are likely mediators of the hypertrophy-sensing function.
734
But even a highly targeted transgenesis may cause problems, as mechanosensors may
735
already be essential for normal muscle function. This is a major challenge for researchers in
736
this area. Another strategy to identify the hypertrophy sensor is based on the knowledge that
737
any hypertrophy sensing protein must physically interact with the proteins that mediate
738
hypertrophy further downstream. Here, interaction proteomic studies in resting and
739
resistance trained skeletal muscle could provide some answers (3). For example,
740
researchers could co-immunoprecipitate mTORC1 protein complexes in resting and
741
resistance exercise-trained muscle to see via mass spectrometry analysis what proteins
742
interact with mTORC1 under load or metabolic stress when compared to rest. This might
743
reveal either the hypertrophy sensor itself or intermediate proteins that connect a
744
hypertrophy sensor to mTORC1 and other downstream hypertrophy mediators. Whilst this
745
sounds feasible, it will be a difficult experiment in reality as the interpretation of interaction
746
proteomic experiments is typically hampered by many false positive results.
747
748
In summary, conclusively identifying major hypertrophy stimuli and their sensors is clearly
749
one of the big remaining questions in exercise physiology. However, experimentally this is
750
difficult to achieve, which explains why there is still a large amount of uncertainty despite
751
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22
many studies. We hope that this review helps to update on the status quo and to stimulate
752
future research in this area.
753
754
Acknowledgements
755
We developed the idea for this review during the International Symposium on Exercise
756
Physiology that took place from the 11-13th of October 2017 at the University of Jyväskylä,
757
Finland. We thank Professor Jari Ylänne, Professor Ju Chen and Dr Matt Alexander for
758
helpful advice.
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760
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40
Figure legends
1275
1276
Figure 1. Schematic overview over how Filamin-C and Bag3 might trigger muscle
1277
hypertrophy in response to resistance exercise (see text for references). 1 Filamin is a Z-
1278
disc-linked protein that binds to actin and becomes deformed in response to mechanical load.
1279
2 Filamin is linked to Bag3 and both Filamin and Bag3 become phosphorylated by unknown
1280
kinases during intense muscle contractions. 3 Bag3 has a WW domain through which is can
1281
bind and sequester proteins with proline-rich PPXY domains including Tsc1, a mTORC1
1282
inhibitor. 4 Bag3 can also sequester inhibitors of the Hippo effector Yap such as Lats1,
1283
Amotl1, and Amotl2. Alternatively, YAP might be important into myonuclei as a result of
1284
nuclear deformation as has been demonstrated in non-muscle cells. Such Yap activation
1285
could be relevant for hypertrophy as YAP can induce the gene that encodes the Lat1 leucine
1286
transporter. 5 Finally, Bag3 also binds to Synpo2 which regulates chaperone-assisted
1287
selective autophaghy (CASA) which regulates the degradation of damaged Z-disc proteins.
1288
1289
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Filamin
(force sensor)
BAG3
WW
Chaperone-assisted selective autophagy
(CASA)
TSC1
PPXY
SYNPO2
PPXY
LATS1
PPXY
mTORC1
YAP LAT1
Protein
synthesis
(also Amotl1 & Amotl2)
P
P
12
3
4
5
Increased branched-chain
amino acid uptake
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