Costameres are mechanosensory sites of focal adhesion in the sarcolemma that reinforce the muscle-fiber composite and provide an anchor for myofibrillogenesis. We hypothesized that elevated content of the integrin-associated regulator of costamere turnover in culture, focal adhesion kinase (FAK), drives changes in costamere component content in antigravity muscle in a load-dependent way in correspondence with altered muscle weight. The content of FAK in soleus muscle being phosphorylated at autoregulatory tyrosine 397 (FAK-pY397) was increased after 20 s of stretch. FAK-pY397 content remained elevated after 24 h of stretch-overload due to upregulated FAK content. Overexpression of FAK in soleus muscle fibers by means of gene electrotransfer increased the β1-integrin (+56%) and meta-vinculin (+88%) content. α7-Integrin (P = 0.46) and γ-vinculin (P = 0.18) content was not altered after FAK overexpression. Co-overexpression of the FAK inhibitor FAK-related nonkinase (FRNK) reduced FAK-pY397 content by 33% and increased the percentage of fast-type fibers that arose in connection with hybrid fibers with gene transfer. Transplantation experiments confirmed the association of FRNK expression with slow-to-fast fiber transformation. Seven days of unloading blunted the elevation of FAK-pY397, β1-integrin, and meta-vinculin content with FAK overexpression, and this was reversed by 1 day of reloading. The results highlight that the expression of components for costameric attachment sites of myofibrils is under load- and fiber type-related control via FAK and its inhibitor FRNK.

costamere components 3 4 Stephan Klossner, Ruowei Li, Severin Ruoss, Anne-Cécile Durieux, Martin 5 Flück 6 7 8 Affiliation: Department of Anatomy, University of Berne, Berne, Switzerland 9 Institute for Biomedical Research into Human Movement and Health, Manchester 10 Metropolitan University, Manchester, United Kingdom 11 Department of Cardiovascular Surgery, University Hospital of Berne, Switzerland 12 13 : corresponding author 14 Institute for Biomedical Research into Human Movement and Health 15 School for HealthCare Science 16 Manchester Metropolitan University 17 Oxford Road 18 Manchester M1 5GD 19 United Kingdom 20 Tel: 0044 161 247 5417 21 Fax: 0044 161 247 5457 22 email: m.flueck@mmu.ac.uk 23 24 25 26 27 28 29 30 31 Running title: Costameres during load-dependent muscle plasticity 32 33 Articles in PresS. Am J Physiol Regul Integr Comp Physiol (July 31, 2013). doi:10.1152/ajpregu.00007.2013

We hypothesized that sarcolemmal adhesion sites of muscle fibres (costameres) are implicated in the load‐dependent regulation of p70S6K signalling towards protein synthesis. Overexpression of focal adhesion kinase (FAK) in rat soleus muscle upregulated the content of the costamere components, gamma‐ and meta‐ vinculin (+153%), p70S6K protein (+40%), and ribosomal transcripts. The expression of p70S6K (−83%), gamma‐ (−39%) and meta‐vinculin (−49%) was reduced with hindlimb unloading and raised again within 24 hours of reloading (p70S6K: 10‐fold; vinculin: 1.5‐fold) in corresponded to FAK activation by Y397 phosphorylation. Similarly, 24 hours of soleus muscle overload increased the content and activation status of FAK and p70S6K. 1 hour after overload the phosphorylation of p70S6K, AKT and 4E‐BP1 was elevated. At this time‐point the content of activated FAK was reduced but was reestablished with co‐overexpression of FAK and its competitor, FRNK, and co‐related with p70S6K content (r=0.74). Changes in muscle loading to human vastus lateralis muscle by bedrest and resistance training reproduced the regulatory relationships between FAK, FAK‐p397 and meta‐vinculin. The observations indicate that remodelling of costameres by FAK is coupled to the load‐dependent control of protein synthetic capacity in vertebrate muscle.

Reprogramming of gene expression contributes to structural and functional adaptation of muscle tissue in response to altered use. The aim of this study was to investigate mechanisms for observed improvements in leg extension strength, gain in relative thigh muscle mass and loss of body and thigh fat content in response to eccentric and conventional strength training in elderly men (n = 14) and women (n = 14; average age of the men and women: 80.1 ± 3.7 years) by means of structural and molecular analyses. Biopsies were collected from m. vastus lateralis in the resting state before and after 12 weeks of training with two weekly resistance exercise sessions (RET) or eccentric ergometer sessions (EET). Gene expression was analyzed using custom-designed low-density PCR arrays. Muscle ultrastructure was evaluated using EM morphometry. Gain in thigh muscle mass was paralleled by an increase in muscle fiber cross-sectional area (hypertrophy) with RET but not with EET, where muscle growth is likely occurring by the addition of sarcomeres in series or by hyperplasia. The expression of transcripts encoding factors involved in muscle growth, repair and remodeling (e.g., IGF-1, HGF, MYOG, MYH3) was increased to a larger extent after EET than RET. MicroRNA 1 expression was decreased independent of the training modality, and was paralleled by an increased expression of IGF-1 representing a potential target. IGF-1 is a potent promoter of muscle growth, and its regulation by microRNA 1 may have contributed to the gain of muscle mass observed in our subjects. EET depressed genes encoding mitochondrial and metabolic transcripts. The changes of several metabolic and mitochondrial transcripts correlated significantly with changes in mitochondrial volume density. Intramyocellular lipid content was decreased after EET concomitantly with total body fat. Changes in intramyocellular lipid content correlated with changes in body fat content with both RET and EET. In the elderly, RET and EET lead to distinct molecular and structural adaptations which might contribute to the observed small quantitative differences in functional tests and body composition parameters. EET seems to be particularly convenient for the elderly with regard to improvements in body composition and strength but at the expense of reducing muscular oxidative capacity.

Stephan Klossner1, Marie-Noelle Giraud2, Sara Sancho Oliver3, David Vaughan4 and Martin Fluck1,4 1Institute of Anatomy, University of Berne, Berne, 2Department of Cardiovascular Surgery, Inselspital, Berne, 3Department of Medicine, University of Fribourg, Fribourg, 4Institute for Biomedical Research into Human Movement and Health, Manchester Metropolitan University, Manchester, 1,2,3Switzerland 4United Kingdom

Striated muscle exhibits a pronounced structural–functional plasticity in response to chronic alterations in loading. We assessed the implication of focal adhesion kinase (FAK) signalling in mechano‐regulated differentiation of slow‐oxidative muscle. Load‐dependent consequences of FAK signal modulation were identified using a multi‐level approach after electrotransfer of rat soleus muscle with FAK‐expression plasmid vs. empty plasmid‐transfected contralateral controls. Muscle fibre‐targeted over‐expression of FAK in anti‐gravitational muscle for 9 days up‐regulated transcript levels of gene ontologies underpinning mitochondrial metabolism and contraction in the transfected belly portion. Concomitantly, mRNA expression of the major fast‐type myosin heavy chain (MHC) isoform, MHC2A, was reduced. The promotion of the slow‐oxidative expression programme by FAK was abolished after co‐expression of the FAK inhibitor FAK‐related non‐kinase (FRNK). Elevated protein content of MHC1 (+9%) and proteins of mitochondrial respiration (+165–610%) with FAK overexpression demonstrated the translation of transcript differentiation in targeted muscle fibres towards a slow‐oxidative muscle phenotype. Coincidentally MHC2A protein was reduced by 50% due to protection of muscle from de‐differentiation with electrotransfer. Fibre cross section in FAK‐transfected muscle was elevated by 6%. The FAK‐modulated muscle transcriptome was load‐dependent and regulated in correspondence to tyrosine 397 phosphorylation of FAK. In the context of overload, the FAK‐induced gene expression became manifest at the level of contraction by a slow transformation and the re‐establishment of normal muscle force from the lowered levels with transfection. These results highlight the analytic power of a systematic somatic transgene approach by mapping a role of FAK in the dominant mechano‐regulation of muscular motor performance via control of gene expression.

We examined the involvement of focal adhesion kinase (FAK) in mechano-regulated signalling to protein synthesis by combining muscle-targeted transgenesis with a physiological model for un- and reloading of hindlimbs. Transfections of mouse tibialis anterior muscle with a FAK expression construct increased FAK protein 1.6-fold versus empty transfection in the contralateral leg and elevated FAK concentration at the sarcolemma. Altered activation status of phosphotransfer enzymes and downstream translation factors showed that FAK overexpression was functionally important. FAK auto-phosphorylation on Y397 was enhanced between 1 and 6 h of reloading and preceded the activation of p70S6K after 24 h of reloading. Akt and translation initiation factors 4E-BP1 and 2A, which reside up- or downstream of p70S6K, respectively, showed no FAK-modulated regulation. The findings identify FAK as an upstream element of the mechano-sensory pathway of p70S6K activation whose Akt-independent regulation intervenes in control of muscle mass by mechanical stimuli in humans.

Sarcopenia is the age-related loss of muscle mass and strength and has been associated with an increased risk of falling and the development of metabolic diseases. Various training protocols, nutritional and hormonal interventions have been proposed to prevent sarcopenia. This study explores the potential of continuous eccentric exercise to retard age-related loss of muscle mass and function. Elderly men and women (80.6 +/- 3.5 years) were randomized to one of three training interventions demanding a training effort of two sessions weekly for 12 weeks: cognitive training (CT; n = 16), conventional resistance training (RET; n = 23) and eccentric ergometer training (EET; n = 23). Subjects were tested for functional parameters and body composition. Biopsies were collected from M. vastus lateralis before and after the intervention for the assessment of fiber size and composition. Maximal isometric leg extension strength (MEL: +8.4 +/- 1.7%) and eccentric muscle coordination (COORD: -43 +/- 4%) were significantly improved with EET but not with RET (MEL: +2.3 +/- 2.0%; COORD: -13 +/- 3%) and CT (MEL: -2.3 +/- 2.5%; COORD: -12 +/- 5%), respectively. We observed a loss of body fat (-5.0 +/- 1.1%) and thigh fat (-6.9 +/- 1.5%) in EET subjects only. Relative thigh lean mass increased with EET (+2.5 +/- 0.6%) and RET (+2.0 +/- 0.3%) and correlated negatively with type IIX/type II muscle fiber ratios. It was concluded that both RET and EET are beneficial for the elderly with regard to muscle functional and structural improvements but differ in their spectrum of effects. A training frequency of only two sessions per week seems to be the lower limit for a training stimulus to reveal measurable benefits.

The present report tested the hypothesis that focal adhesion kinase (FAK) is a myocellular transducer of mechanical signals towards downstream control of slow, fatigue‐resistant muscle fibres. This examination was approached in an integrative setting combining somatic transgenesis for myocellular FAK overexpression with transcript profiling, morphometry and myography in two physiological rat models for load‐dependent plasticity of anti‐gravitational soleus muscle. The molecular measures identified FAK‐regulated expressional reprogramming of gene ontologies underlying the oxidative and mechanical specialisation of soleus muscle. FAK‐dependent transcript levels were suppressed with unloading but transient elevated along with muscle weight with reloading. The transcript upregulation of energy and protein metabolism with reloading corresponded to increased tyrosine 397 phosphorylation and the translocation of FAK to the sarcolemma. The load‐dependent modulation of the soleus muscle phenotype by FAK was related to fibre regeneration and became manifest as a shift towards slow contractile characteristics with functional overload. Collectively the findings expose spatially controlled FAK signalling as a key upstream element of the mechano‐dependent muscle differentiation.

Tenascin-C (TNC) is a mechano-regulated, morphogenic, extracellular matrix protein that is associated with tissue remodeling. The physiological role of TNC remains unclear because transgenic mice engineered for a TNC deficiency, via a defect in TNC secretion, show no major pathologies. We hypothesized that TNC-deficient mice would demonstrate defects in the repair of damaged leg muscles, which would be of functional significance because this tissue is subjected to frequent cycles of mechanical damage and regeneration. TNC-deficient mice demonstrated a blunted expression of the large TNC isoform and a selective atrophy of fast-muscle fibers associated with a defective, fast myogenic expression response to a damaging mechanical challenge. Transcript profiling mapped a set of de-adhesion, angiogenesis, and wound healing regulators as TNC expression targets in striated muscle. Expression of these regulators correlated with the residual expression of a damage-related 200-kDa protein, which resembled the small TNC isoform. Somatic knockin of TNC in fast-muscle fibers confirmed the activation of a complex expression program of interstitial and slow myofiber repair by myofiber-derived TNC. The results presented here show that a TNC-orchestrated molecular pathway integrates muscle repair into the load-dependent control of the striated muscle phenotype.

Tenascin-C (TNC) is a mechano-regulated, morphogenic, extracellular matrix protein which is associated with tissue remodeling. The physiological role of TNC remains unclear because transgenic mice engineered for a TNC-deficiency via a defect in TNC secretion show no major pathologies. We hypothesized that TNC-deficient mice would demonstrate defects in the repair of damaged leg muscles, which would be of functional significance because this tissue is subjected to frequent cycles of mechanical damage and regeneration. TNC-deficient mice demonstrated a blunted expression of the large TNC isoform, and a selective atrophy of fast muscle fibers, associated with a defective fast myogenic expression response to a damaging mechanical challenge. Transcript profiling mapped a set of de-adhesion, angiogenesis, and wound healing regulators as TNC expression targets in striated muscle. Expression of these regulators correlated with the residual expression of a damage-related 200-kDa protein, which resembled the small TNC isoform. Somatic Knock-in of TNC in fast muscle fibers confirmed the activation of a complex expression program of interstitial and slow myofiber repair by myofiber-derived TNC. The results presented here show for the first time that a TNC-orchestrated molecular pathway integrates muscle repair into the load-dependent control of the striated muscle phenotype.

The number of elderly people is growing in western populations, but only few maximal performance data exist for people >75 years, in particular for European octogenarians. This study was performed to characterize maximal performance of 55 independently living subjects (32 women, 81.1 +/- 3.4 years; 23 men, 81.7 +/- 2.9 years) with a focus on sex differences. Maximal performance was determined in a ramp test to exhaustion on a bicycle ergometer with ergospirometry, electrocardiogram and blood lactate measurements. Maximal isometric extension strength of the legs (MEL) was measured on a force platform in a seated position. Body composition was quantified by X-ray absorptiometry. In >25% of the subjects, serious cardiac abnormalities were detected during the ramp test with men more frequently being affected than women. Maximal oxygen consumption and power output were 18.2 +/- 3.2 versus 25.9 +/- 5.9 ml min(-1) kg(-1) and 66 +/- 12 versus 138 +/- 40 W for women versus men, with a significant sex difference for both parameters. Men outperformed women for MEL with 19.0 +/- 3.8 versus 13.6 +/- 3.3 N kg(-1). Concomitantly, we found a higher proportion of whole body fat in women (32.1 +/- 6.2%) compared to men (20.5 +/- 4.4%). Our study extends previously available maximal performance data for endurance and strength to independently living European octogenarians. As all sex-related differences were still apparent after normalization to lean body mass, it is concluded that it is essential to differentiate between female and male subjects when considering maximal performance parameters in the oldest segment of our population.

The muscle has a wide range of possibilities to adapt its phenotype. Repetitive submaximal concentric exercise (i.e., shortening contractions) mainly leads to adaptations of muscle oxidative metabolism and endurance while eccentric exercise (i.e., lengthening contractions) results in muscle growth and gain of muscle strength. Modified gene expression is believed to mediate these exercise-specific muscle adjustments. In the present study, early alterations of the gene expression signature were monitored by a muscle-specific microarray. Transcript profiling was performed on muscle biopsies of vastus lateralis obtained from six male subjects before and in a 24-h time course after a single bout of mild eccentric ergometer exercise. The eccentric exercise consisted of 15 min of eccentric cycling at 50% of the individual maximal concentric power output leading to muscle soreness (5.9 on a 0–10 visual analogue scale) and limited muscle damage (1.7-fold elevated creatine kinase activity). Muscle impairment was highlighted by a transient reduction in jumping height after the eccentric exercise. On the gene expression level, we observed a general early downregulation of detected transcripts, followed by a slow recovery close to the control values within the first 24 h post exercise. Only very few regulatory factors were increased. This expression signature is different from the signature of a previously published metabolic response after an intensive endurance-type concentric exercise as well as after maximal eccentric exercise. This is the first description of the time course of changes in gene expression as a consequence of a mild eccentric stimulus.

A number of molecular tools enable us to study the mechanisms of muscle plasticity. Ideally, this research is conducted in view of the structural and functional consequences of the exercise-induced changes in gene expression. Muscle cells are able to detect mechanical, metabolic, neuronal and hormonal signals which are transduced over multiple pathways to the muscle genome. Exercise activates many signaling cascades - the individual characteristic of the stress leading to a specific response of a network of signaling pathways. Signaling typically results in the transcription of multiple early genes among those of the well known fos and jun family, as well as many other transcription factors. These bind to the promoter regions of downstream genes initiating the structural response of muscle tissue. While signaling is a matter of minutes, early genes are activated over hours leading to a second wave of transcript adjustments of structure genes that can then be effective over days. Repeated exercise sessions thus lead to a concerted accretion of mRNAs which upon translation results in a corresponding protein accretion. On the structural level, the protein accretion manifests itself for instance as an increase in mitochondrial volume upon endurance training or an increase in myofibrillar proteins upon strength training. A single exercise stimulus carries a molecular signature which is typical both for the type of stimulus (i.e. endurance vs. strength) as well as the actual condition of muscle tissue (i.e. untrained vs. trained). Likewise, it is clearly possible to distinguish a molecular signature of an expressional adaptation when hypoxic stress is added to a regular endurance exercise protocol in well-trained endurance athletes. It therefore seems feasible to use molecular tools to judge the properties of an exercise stimulus much earlier and at a finer level than is possible with conventional functional or structural techniques.