nNOS Increases Fiber Type-Specific Angiogenesis in Skeletal Muscle of Mice in Response to Endurance Exercise.

We studied the relationship between neuronal NO synthase (nNOS) expression and capillarity in the tibialis anterior (TA) muscle of mice subjected to treadmill training. The mRNA (+131%) and protein (+63%) levels of nNOS were higher (p ≤ 0.05) in the TA muscle of C57BL/6 mice undergoing treadmill training for 28 days than in those of littermates remaining sedentary, indicating an up-regulation of nNOS by endurance exercise. Both TA muscles of 16 C57BL/6 mice were subjected to gene electroporation with either the pIRES2-ZsGreen1 plasmid (control plasmid) or the pIRES2-ZsGreen1-nNOS gene-inserted plasmid (nNOS plasmid). Subsequently, one group of mice (n = 8) underwent treadmill training for seven days, while the second group of mice (n = 8) remained sedentary. At study end, 12-18% of TA muscle fibers expressed the fluorescent reporter gene ZsGreen1. Immunofluorescence for nNOS was 23% higher (p ≤ 0.05) in ZsGreen1-positive fibers than ZsGreen1-negative fibers from the nNOS-transfected TA muscle of mice subjected to treadmill training. Capillary contacts around myosin heavy-chain (MHC)-IIb immunoreactive fibers (14.2%; p ≤ 0.05) were only higher in ZsGreen1-positive fibers than ZsGreen1-negative fibers in the nNOS-plasmid-transfected TA muscles of trained mice. Our observations are in line with an angiogenic effect of quantitative increases in nNOS expression, specifically in type-IIb muscle fibers after treadmill training.

Down-Regulation of Mitochondrial Metabolism after Tendon Release Primes Lipid Accumulation in Rotator Cuff Muscle.

Atrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of ß-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration.

Recovery from 6-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting.

The Sarcolab pilot study of 2 crewmembers, investigated before and after a 6-mo International Space Station mission, has demonstrated the substantial muscle wasting and weakness, along with disruption of muscle's oxidative metabolism. The present work aimed at evaluating the pro/anti-inflammatory status in the same 2 crewmembers (A, B). Blood circulating (c-)microRNAs (miRs), c-proteasome, c-mitochondrial DNA, and cytokines were assessed by real-time quantitative PCR or ELISA tests. Time series analysis was performed ( i.e., before flight and after landing) at 1 and 15 d of recovery (R+1 and R+15, respectively). C-biomarkers were compared with an age-matched control population and with 2-dimensional proteomic analysis of the 2 crewmembers' muscle biopsies. Striking differences were observed between the 2 crewmembers at R+1, in terms of inflamma-miRs (c-miRs-21-5p, -126-3p, and -146a-5p), muscle specific (myo)-miR-206, c-proteasome, and IL-6/leptin, thus making the 2 astronauts dissimilar to each other. Final recovery levels of c-proteasome, c-inflamma-miRs, and c-myo-miR-206 were not reverted to the baseline values in crewmember A. In both crewmembers, myo-miR-206 changed significantly after recovery. Muscle biopsy of astronaut A showed an impressive 80% increase of α-1-antitrypsin, a target of miR-126-3p. These results point to a strong stress response induced by spaceflight involving muscle tissue and the proinflammatory setting, where inflamma-miRs and myo-miR-206 mediate the systemic recovery phase after landing.-Capri, M., Morsiani, C., Santoro, A., Moriggi, M., Conte, M., Martucci, M., Bellavista, E., Fabbri, C., Giampieri, E., Albracht, K., Flück, M., Ruoss, S., Brocca, L., Canepari, M., Longa, E., Di Giulio, I., Bottinelli, R., Cerretelli, P., Salvioli, S., Gelfi, C., Franceschi, C., Narici, M., Rittweger, J. Recovery from 6-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting.

The Cardiovascular Response to Interval Exercise Is Modified by the Contraction Type and Training in Proportion to Metabolic Stress of Recruited Muscle Groups.

BACKGROUND: Conventional forms of endurance training based on shortening contractions improve aerobic capacity but elicit a detriment of muscle strength. We hypothesized that eccentric interval training, loading muscle during the lengthening phase of contraction, overcome this interference and potentially adverse cardiovascular reactions, enhancing both muscle metabolism and strength, in association with the stress experienced during exercise. METHODS: Twelve healthy participants completed an eight-week program of work-matched progressive interval-type pedaling exercise on a soft robot under predominately concentric or eccentric load. RESULTS: Eccentric interval training specifically enhanced the peak power of positive anaerobic contractions (+28%), mitigated the strain on muscle's aerobic metabolism, and lowered hemodynamic stress during interval exercise, concomitant with a lowered contribution of positive work to the target output. Concentric training alone lowered blood glucose concentration during interval exercise and mitigated heart rate and blood lactate concentration during ramp exercise. Training-induced adjustments for lactate and positive peak power were independently correlated (p < 0.05, |r| > 0.7) with indices of metabolic and mechanical muscle stress during exercise. DISCUSSION: Task-specific improvements in strength and muscle's metabolic capacity were induced with eccentric interval exercise lowering cardiovascular risk factors, except for blood glucose concentration, possibly through altered neuromuscular coordination.

Neurectomy preserves fast fibers when combined with tenotomy of infraspinatus muscle via upregulation of myogenesis.

INTRODUCTION: We evaluated the contribution of denervation-related molecular processes to rotator cuff muscle degeneration after tendon release. METHODS: We assessed the levels of myogenic (myogenin and myogenic differentiation factor [myoD]) and proadipogenic (peroxisome proliferator-activated receptor γ) transcription factors; the denervation-associated proteins tenascin-C, laminin-2, and calcium/calmodulin-dependent kinase II (CaMKII); and cellular alterations in sheep after infraspinatus tenotomy (TEN), suprascapular neurectomy (NEU), or both (TEN-NEU). RESULTS: Extracellular ground substance increased at the expense of contractile tissue 16 weeks after surgery, correlating with CaMKII isoform levels. Sheep undergoing NEU and TEN-NEU had exaggerated infraspinatus atrophy and increased fast fibers compared with TEN sheep. The ßMCaMKII isoform levels increased with TEN, and myoD levels tripled after denervation and were associated with slow fibers. DISCUSSION: In sheep, denervation did not affect muscle-to-fat conversion after TEN of the infraspinatus. Furthermore, concurrent NEU mitigated the loss of fast fibers after TEN by inducing a fast-contractile phenotype. Muscle Nerve 59:100-107, 2019.

Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction.

Achilles tendon rupture necessitates rapid tendon reattachment to reinstate plantar flexion before affected muscles deteriorate through muscle fiber atrophy and transformation. The implicated process may involve alterations in sarcolemmal sites of myofibril attachment (costameres), which control myofibrillogenesis via a mechano-regulated mechanism through integrin-associated focal adhesion kinase (FAK). We assessed the contribution of FAK to alterations in fiber type composition and expression of costamere-associated structural proteins, the phosphorylation status of Y397-FAK and downstream mTOR/JNK-P70S6K hypertrophy signaling in rat soleus muscle after Achilles tenotomy and tendon repair. Achilles tenotomy induced a profound deterioration of muscle composition 14 days, but not 4 days, following tendon release, comprising specifically increased area percentages of fast type fibers, fibers with internal nuclei, and connective tissue. Concomitantly, expression of costameric proteins FAK and meta-vinculin, and phosphorylation of T421/S424-P70S6K and T183/Y185-JNK was elevated, all of which was mitigated by tendon reattachment immediately after release. Overexpression of FAK in soleus muscle fibers and reattachment corrected the expression of meta- and gamma-vinculin isoforms to the lower levels in mock controls while further enhancing T183/Y185-JNK phosphorylation and levels of FAK C-terminus-related inhibitory proteins. Co-overexpression of the FAK inhibitor, FRNK, lowered FAK-overexpression driven Y397-FAK phosphorylation and T183/Y185-JNK phosphorylation. FAK levels correlated to molecular and cellular hallmarks of fiber degeneration. The findings demarcate the window between 4 and 14 days after tenotomy as costamere-dependent muscle transformation process, and expose that FAK overexpression prevents molecular aspects of the pathology which within the study limitations does not result in the mitigation of muscle fiber degeneration.250 words.

Delayed-Onset Muscle Soreness: Temporal Assessment With Quantitative MRI and Shear-Wave Ultrasound Elastography.

OBJECTIVE: The objective of our study was to assess delayed-onset muscle soreness (DOMS) over time using quantitative MRI and shear-wave ultrasound (US) elastography. SUBJECTS AND METHODS: Five male (mean age ± SD, 39.6 ± 4.6 years) and five female (30.6 ± 13.5 years) volunteers underwent 1.5-T MRI before and after (15 minutes, 1 day, 3 days, 7 days) performing unilateral eccentric resistance exercise of the elbow flexor muscles. The MRI examinations included fluid-sensitive, DWI, and diffusion-tensor imaging sequences of the distal upper arm. Muscle edema, apparent diffusion coefficient (ADC), and fractional anisotropy (FA) were assessed. US of the brachialis muscle was performed before and after (15 minutes, 12 hours, 1 day, 2 days, 3 days, 7 days) exercise to measure mean shear-wave velocity (SWV). Pain and muscle tightness were assessed. RESULTS: For men, muscle edema was moderate and peaked 3 days after exercise; for women, muscle edema was mild and peaked 1-3 days after exercise. ADC was highest 3 days after exercise in men (mean, 1809.22 × 10-6 mm2/s; before exercise, 1529.88 × 10-6 mm2/s) and women (1741.90 × 10-6 mm2/s; before exercise, 1475.80 × 10-6 mm2/s). FA dropped from 361.00 in men and 389.00 in women before exercise to a minimum of 252.12 and 321.28, respectively, 3 days after exercise. Mean SWV increased after exercise in men (before exercise, 3.00 ± 0.30 m/s; peak [15 minutes after exercise], 4.04 ± 0.90 m/s) and women (before, 2.82 ± 0.40 m/s; peak [1 day after exercise], 3.23 ± 0.40 m/s) and subsequently returned to normal. In men, the ADC values of the brachialis muscle positively correlated with mean SWV (r = 0.92, p = 0.028). FA negatively correlated with pain in men (r = -0.993, p = 0.001) Muscle edema outlasted clinical symptoms in most volunteers. CONCLUSION: FA inversely correlates with pain and may be a useful imaging parameter for assessment of DOMS. Shear-wave US elastography shows a temporary increase of muscle stiffness after DOMS-inducing exercise but does not correlate with quantitative MRI parameters or clinical symptoms.

Robot-assisted assessment of muscle strength.

Impairment of neuromuscular function in neurological disorders leads to reductions in muscle force, which may lower quality of life. Rehabilitation robots that are equipped with sensors are able to quantify the extent of muscle force impairment and to monitor a patient during the process of neurorehabilitation with sensitive and objective assessment methods. In this article, we provide an overview of fundamental aspects of muscle function and how the corresponding variables can be quantified by means of meaningful robotic assessments that are primarily oriented towards upper limb neurorehabilitation. We discuss new concepts for the assessment of muscle function, and present an overview of the currently available systems for upper limb measurements. These considerations culminate in practical recommendations and caveats for the rational quantification of force magnitude, force direction, moment of a force, impulse, critical force (neuromuscular fatigue threshold) and state and trait levels of fatigue.

Quantitative Shear-Wave US Elastography of the Supraspinatus Muscle: Reliability of the Method and Relation to Tendon Integrity and Muscle Quality.

PURPOSE: To evaluate the reliability of ultrasonographic (US) elastography of the supraspinatus (SSP) muscle, define normal shear-wave velocity (SWV) values, and correlate findings with tendon integrity and muscle quality. MATERIALS AND METHODS: The study was approved by the local ethics committee, and written informed consent was obtained from all patients. SSP SWV (in meters per second) was prospectively assessed twice in 22 asymptomatic volunteers (mean age ± standard deviation, 53.8 years ± 15.3; 11 women and 11 men) by two independent examiners by using shear-wave elastography. Forty-four patients (mean age, 51.9 years ± 15.0; 22 women and 22 men) were prospectively included. SWV findings were compared with tendon integrity, tendon retraction (Patte classification), fatty muscle infiltration (Goutallier stages 0-IV), and muscle volume atrophy (tangent sign) on magnetic resonance (MR) images. Descriptive statistics, Spearman correlation, analysis of variance, two-sample t test, and intraclass correlation coefficient (ICC) were used. RESULTS: Test-retest reliability for mean total SWV (MTSWV) was good for examiner 1 (ICC = 0.70; 95% confidence interval [CI]: 0.30, 0.87; P = .003) and excellent for examiner 2 (ICC = 0.80; 95% CI: 0.53, 0.92; P < .001). Interexaminer reliability was excellent (ICC = 0.89; 95% CI: 0.64, 0.96; P < .001). MTSWV in volunteers (3.0 m/sec ± 0.5) was significantly higher than that in patients (2.5 m/sec ± 0.5; P = .001). For tendon integrity, no significant difference in MTSWV was found. For tendon retraction, MTSWV varies significantly between patients with different degrees of retraction (P = .047). No significant differences were found for Goutallier subgroups. MTSWV was significantly lower with a positive tangent sign (P = .015; n = 10). CONCLUSION: Shear-wave elastography is reproducible for assessment of the SSP muscle. Mean normal SSP SWV is 3.0 m/sec ± 0.5. SWV decreases with increasing fat content (Goutallier stage 0-III) and increases in the final stage of fatty infiltration (Goutallier stage IV).

One bout of vibration exercise with vascular occlusion activates satellite cells.

NEW FINDINGS: What is the central question of this study? Acute skeletal muscle satellite cell (SC) activation is associated with skeletal muscle hypertrophy. Although the quantity of SCs has been reported to increase following a single bout of resistance exercise, data on muscle fibre type-specific SC quantity and/or activation status after a single bout of vibration is presently lacking. What is the main finding and its importance? By determining SCs from muscle biopsies of the vastus lateralis using immunohistochemistry, we conclude that modification of vibration exercise by superimposition of occlusion induced activation and differentiation of SCs in young men, which had not been observed with whole-body vibration or blood flow restriction alone. We tested the hypothesis that whole-body vibration (WBV) is insufficient to expand satellite cell numbers 24 h postexercise, whereas WBV in combination with blood flow restriction (BFR) is sufficient. Twenty-five young men were randomly assigned to one of the following three groups: WBV, BFR exercise or WBVBFR. Satellite cell numbers were determined from muscle biopsies of the vastus lateralis muscle using immunohistochemistry. Satellite cell quantity and frequency (+99.4%, P = 0.012 and +77.1%, P = 0.010, respectively) increased only in the WBVBFR group. Similar results were obtained for the quantity and frequency of myogenin-positive myonuclei (+139.0%, P < 0.001 and +148.4%, P < 0.001, respectively). We conclude that modification of WBV by superimposition of BFR induced activation and differentiation of satellite cells in young men, which had not been observed with WBV or BFR alone. These data suggest that WBVBFR might represent a novel viable anabolic stimulus.

CaMKII content affects contractile, but not mitochondrial, characteristics in regenerating skeletal muscle.

BACKGROUND: The multi-meric calcium/calmodulin-dependent protein kinase II (CaMKII) is the main CaMK in skeletal muscle and its expression increases with endurance training. CaMK family members are implicated in contraction-induced regulation of calcium handling, fast myosin type IIA expression and mitochondrial biogenesis. The objective of this study was to investigate the role of an increased CaMKII content for the expression of the contractile and mitochondrial phenotype in vivo. Towards this end we attempted to co-express alpha- and beta-CaMKII isoforms in skeletal muscle and characterised the effect on the contractile and mitochondrial phenotype. RESULTS: Fast-twitch muscle m. gastrocnemius (GM) and slow-twitch muscle m. soleus (SOL) of the right leg of 3-month old rats were transfected via electro-transfer of injected expression plasmids for native α/ß CaMKII. Effects were identified from the comparison to control-transfected muscles of the contralateral leg and non-transfected muscles. α/ß CaMKII content in muscle fibres was 4-5-fold increased 7 days after transfection. The transfection rate was more pronounced in SOL than GM muscle (i.e. 12.6 vs. 3.5%). The overexpressed α/ß CaMKII was functional as shown through increased threonine 287 phosphorylation of ß-CaMKII after isometric exercise and down-regulated transcripts COXI, COXIV, SDHB after high-intensity exercise in situ. α/ß CaMKII overexpression under normal cage activity accelerated excitation-contraction coupling and relaxation in SOL muscle in association with increased SERCA2, ANXV and fast myosin type IIA/X content but did not affect mitochondrial protein content. These effects were observed on a background of regenerating muscle fibres. CONCLUSION: Elevated CaMKII content promotes a slow-to-fast type fibre shift in regenerating muscle but is not sufficient to stimulate mitochondrial biogenesis in the absence of an endurance stimulus.

Hypoxia refines plasticity of mitochondrial respiration to repeated muscle work.

PURPOSE: We explored whether altered expression of factors tuning mitochondrial metabolism contributes to muscular adaptations with endurance training in the condition of lowered ambient oxygen concentration (hypoxia) and whether these adaptations relate to oxygen transfer as reflected by subsarcolemmal mitochondria and oxygen metabolism in muscle. METHODS: Male volunteers completed 30 bicycle exercise sessions in normoxia or normobaric hypoxia (4,000 m above sea level) at 65% of the respective peak aerobic power output. Myoglobin content, basal oxygen consumption, and re-oxygenation rates upon reperfusion after 8 min of arterial occlusion were measured in vastus muscles by magnetic resonance spectroscopy. Biopsies from vastus lateralis muscle, collected pre and post a single exercise bout, and training, were assessed for levels of transcripts and proteins being associated with mitochondrial metabolism. RESULTS: Hypoxia specifically lowered the training-induced expression of markers of respiratory complex II and IV (i.e. SDHA and isoform 1 of COX-4; COX4I1) and preserved fibre cross-sectional area. Concomitantly, trends (p < 0.10) were found for a hypoxia-specific reduction in the basal oxygen consumption rate, and improvements in oxygen repletion, and aerobic performance in hypoxia. Repeated exercise in hypoxia promoted the biogenesis of subsarcolemmal mitochondria and this was co-related to expression of isoform 2 of COX-4 with higher oxygen affinity after single exercise, de-oxygenation time and myoglobin content (r ≥ 0.75). Conversely, expression in COX4I1 with training correlated negatively with changes of subsarcolemmal mitochondria (r < -0.82). CONCLUSION: Hypoxia-modulated adjustments of aerobic performance with repeated muscle work are reflected by expressional adaptations within the respiratory chain and modified muscle oxygen metabolism.

Enhanced capacity for CaMKII signaling mitigates calcium release related contractile fatigue with high intensity exercise.

BACKGROUND: We tested whether enhancing the capacity for calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling would delay fatigue of excitation-induced calcium release and improve contractile characteristics of skeletal muscle during fatiguing exercise. METHODS: Fast and slow type muscle, gastrocnemius medialis (GM) and soleus (SOL), of rats and mouse interosseus (IO) muscle fibers, were transfected with pcDNA3-based plasmids for rat α and ß CaMKII or empty controls. Levels of CaMKII, its T287-phosphorylation (pT287-CaMKII), and phosphorylation of components of calcium release and re-uptake, ryanodine receptor 1 (pS2843-RyR1) and phospholamban (pT17-PLN), were quantified biochemically. Sarcoplasmic calcium in transfected muscle fibers was monitored microscopically during trains of electrical excitation based on Fluo-4 FF fluorescence (n = 5-7). Effects of low- (n = 6) and high- (n = 8) intensity exercise on pT287-CaMKII and contractile characteristics were studied in situ. RESULTS: Co-transfection with αCaMKII-pcDNA3/ßCaMKII-pcDNA3 increased α and ßCaMKII levels in SOL (+45.8 %, +250.5 %) and GM (+40.4 %, +89.9 %) muscle fibers compared to control transfection. High-intensity exercise increased pT287-ßCaMKII and pS2843-RyR1 levels in SOL (+269 %, +151 %) and GM (+354 %, +119 %), but decreased pT287-αCaMKII and p17-PLN levels in GM compared to SOL (-76 % vs. +166 %; 0 % vs. +128 %). α/ß CaMKII overexpression attenuated the decline of calcium release in muscle fibers with repeated excitation, and mitigated exercise-induced deterioration of rates in force production, and passive force, in a muscle-dependent manner, in correlation with pS2843-RyR1 and pT17-PLN levels (|r| > 0.7). CONCLUSION: Enhanced capacity for α/ß CaMKII signaling improves fatigue-resistance of active and passive contractile muscle properties in association with RyR1- and PLN-related improvements in sarcoplasmic calcium release.

Poly(ADP-Ribose) Polymerases-Inhibitor Talazoparib Inhibits Muscle Atrophy and Fatty Infiltration in a Tendon Release Infraspinatus Sheep Model: A Pilot Study.

Structural muscle changes, including muscle atrophy and fatty infiltration, follow rotator cuff tendon tear and are associated with a high repair failure rate. Despite extensive research efforts, no pharmacological therapy is available to successfully prevent both muscle atrophy and fatty infiltration after tenotomy of tendomuscular unit without surgical repair. Poly(ADP-ribose) polymerases (PARPs) are identified as a key transcription factors involved in the maintenance of cellular homeostasis. PARP inhibitors have been shown to influence muscle degeneration, including mitochondrial hemostasis, oxidative stress, inflammation and metabolic activity, and reduced degenerative changes in a knockout mouse model. Tenotomized infraspinatus were assessed for muscle degeneration for 16 weeks using a Swiss Alpine sheep model (n = 6). All sheep received daily oral administration of 0.5 mg Talazoparib. Due to animal ethics, the treatment group was compared with three different controls from prior studies of our institution. To mitigate potential batch heterogeneity, PARP-I was evaluated in comparison with three distinct control groups (n = 6 per control group) using the same protocol without treatment. The control sheep were treated with an identical study protocol without Talazoparib treatment. Muscle atrophy and fatty infiltration were evaluated at 0, 6 and 16 weeks post-tenotomy using DIXON-MRI. The controls and PARP-I showed a significant (control p < 0.001, PARP-I p = 0.01) decrease in muscle volume after 6 weeks. However, significantly less (p = 0.01) atrophy was observed in PARP-I after 6 weeks (control 1: 76.6 ± 8.7%; control 2: 80.3 ± 9.3%, control 3: 73.8 ± 6.7% vs. PARP-I: 90.8 ± 5.1% of the original volume) and 16 weeks (control 1: 75.7 ± 9.9; control 2: 74.2 ± 5.6%; control 3: 75.3 ± 7.4% vs. PARP-I 93.3 ± 10.6% of the original volume). All experimental groups exhibited a statistically significant (p < 0.001) augmentation in fatty infiltration following a 16-week period when compared to the initial timepoint. However, the PARP-I showed significantly less fatty infiltration (p < 0.003) compared to all controls (control 1: 55.6 ± 6.7%, control 2: 53.4 ± 9.4%, control 3: 52.0 ± 12.8% vs. PARP-I: 33.5 ± 8.4%). Finally, a significantly (p < 0.04) higher proportion and size of fast myosin heavy chain-II fiber type was observed in the treatment group. This study shows that PARP-inhibition with Talazoparib inhibits the progression of both muscle atrophy and fatty infiltration over 16 weeks in retracted sheep musculotendinous units.

Quantitative changes in focal adhesion kinase and its inhibitor, FRNK, drive load-dependent expression of costamere components.

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 ß1-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, ß1-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 remodeling with muscle loading and unloading in healthy young men.

Costameres are mechano-sensory sites of focal adhesion in the sarcolemma that provide a structural anchor for myofibrils. Their turnover is regulated by integrin-associated focal adhesion kinase (FAK). We hypothesized that changes in content of costamere components (beta 1 integrin, FAK, meta-vinculin, gamma-vinculin) with increased and reduced loading of human anti-gravity muscle would: (i) relate to changes in muscle size and molecular parameters of muscle size regulation [p70S6K, myosin heavy chain (MHC)1 and MHCIIA]; (ii) correspond to adjustments in activity and expression of FAK, and its negative regulator, FRNK; and (iii) reflect the temporal response to reduced and increased loading. Unloading induced a progressive decline in thickness of human vastus lateralis muscle after 8 and 34 days of bedrest (-4% and -14%, respectively; n = 9), contrasting the increase in muscle thickness after 10 and 27 days of resistance training (+5% and +13%; n = 6). Changes in muscle thickness were correlated with changes in cross-sectional area of type I muscle fibers (r = 0.66) and beta 1 integrin content (r = 0.76) at the mid-point of altered loading. Changes in meta-vinculin and FAK-pY397 content were correlated (r = 0.85) and differed, together with the changes of beta 1 integrin, MHCI, MHCII and p70S6K, between the mid- and end-point of resistance training. By contrast, costamere protein level changes did not differ between time points of bedrest. The findings emphasize the role of FAK-regulated costamere turnover in the load-dependent addition and removal of myofibrils, and argue for two phases of muscle remodeling with resistance training, which do not manifest at the macroscopic level.

A mixed-effects model of the dynamic response of muscle gene transcript expression to endurance exercise.

Altered expression of a broad range of gene transcripts after exercise reflects the specific adjustment of skeletal muscle makeup to endurance training. Towards a quantitative understanding of this molecular regulation, we aimed to build a mixed-effects model of the dynamics of co-related transcript responses to exercise. It was built on the assumption that transcript levels after exercise varied because of changes in the balance between transcript synthesis and degradation. It was applied to microarray data of 231 gene transcripts in vastus lateralis muscle of six subjects 1, 8 and 24 h after endurance exercise and 6-week training on a stationary bicycle. Cluster analysis was used to select groups of transcripts having highest co-correlation of their expression (r > 0.70): Group 1 comprised 45 transcripts including factors defining the oxidative and contractile phenotype and Group 2 included 39 transcripts mainly defined by factors found at the cell periphery and the extracellular space. Data from six subjects were pooled to filter experimental noise. The model fitted satisfactorily the responses of Group 1 (r (2) = 0.62 before and 0.85 after training, P < 0.001) and Group 2 (r (2) = 0.75 and 0.79, P < 0.001). Predicted variation in transcription rate induced by exercise yielded a difference in amplitude and time-to-peak response of gene transcripts between the two groups before training and with training in Group 2. The findings illustrate that a mixed-effects model of transcript responses to exercise is suitable to explore the regulation of muscle plasticity by training at the transcriptional level and indicate critical experiments needed to consolidate model parameters empirically.

The angiotensin converting enzyme insertion/deletion polymorphism alters the response of muscle energy supply lines to exercise.

The presence of a silencing sequence (the I-allele) in the gene for the upstream regulator of blood flow, angiotensin I-converting enzyme (ACE), is associated with superior endurance performance and its trainability. We tested in a retrospective study with 36 Caucasian men of Swiss descent whether carriers of the ACE I-allele demonstrate a modified adaptive response of energy supply lines in knee extensor muscle, and aerobic fitness, to endurance training based on 6 weeks of supervised bicycle exercise or 6 months of self-regulated running (p value

ACE-I/D Allele Modulates Improvements of Cardiorespiratory Function and Muscle Performance with Interval-Type Exercise.

Background: The prominent insertion/deletion polymorphism in the gene for the major modulator of tissue perfusion, angiotensin-converting enzyme (ACE-I/D) is associated with variability in adjustments in cardiac and skeletal muscle performance with standard forms of endurance and strength type training. Here, we tested whether the ACE-I/D genotype would be associated with variability in the effects of interval-type training on peak and aerobic performance of peripheral muscle and cardio-vasculature and post-exercise recovery. Methods: Nine healthy subjects (39.0 ± 14.7 years of age; 64.6 ± 16.1 kg, 173.6 ± 9.9) completed eight weeks of interval training on a soft robotic device based on repeated sets of a pedaling exercise at a matched intensity relative to their peak aerobic power output. Prior to and post-training, peak anaerobic and aerobic power output was assessed, mechanical work and metabolic stress (oxygen saturation and hemoglobin concentrations of Musculus vastus lateralis (VAS) and Musculus gastrocnemius (GAS), blood lactate and factors setting cardiac output such as heart rate, systolic and diastolic blood pressure were monitored during ramp-incremental exercise and interval exercise with the calculation of areas under the curve (AUC), which were put in relation to the produced muscle work. Genotyping was performed based on I- and D-allele-specific polymerase chain reactions on genomic DNA from mucosal swaps. The significance of interaction effects between training and ACE I-allele on absolute and work-related values was assessed with repeated measures ANOVA. Results: Subjects delivered 87% more muscle work/power, 106% more cardiac output, and muscles experienced ~72% more of a deficit in oxygen saturation and a ~35% higher passage of total hemoglobin during single interval exercise after the eight weeks of training. Interval training affected aspects of skeletal muscle metabolism and performance, whose variability was associated with the ACE I-allele. This concerned the economically favorable alterations in the work-related AUC for the deficit of SmO2 in the VAS and GAS muscles during the ramp exercise for the I-allele carriers and opposing deteriorations in non-carriers. Conversely, oxygen saturation in the VAS and GAS at rest and during interval exercise was selectively improved after training for the non-carriers of the I-allele when the AUC of tHb per work during interval exercise deteriorated in the carriers. Training also improved aerobic peak power output by 4% in the carriers but not the non-carriers (p = 0.772) of the ACE I-allele while reducing negative peak power (-27.0%) to a lesser extent in the ACE I-allele carriers than the non-carriers. Variability in cardiac parameters (i.e., the AUC of heart rate and glucose during ramp exercise, was similar to the time to recovery of maximal tHb in both muscles after cessation of ramp exercise, only associated with the ACE I-allele but not training per se. Diastolic blood pressure and cardiac output during recovery from exhaustive ramp exercise demonstrated a trend for training-associated differences in association with the ACE I-allele. Discussion: The exercise-type dependent manifestation of antidromic adjustments in leg muscle perfusion and associated local aerobic metabolism between carriers and non-carriers of the ACE I-allele with the interval-training highlight that non-carriers of the I-allele do not present an essential handicap to improve perfusion-related aerobic muscle metabolism but that the manifestation of responsiveness depends on the produced work. Conclusions: The deployed interval-type of exercise produced ACE I-allele-related differences in the alterations of negative anaerobic performance and perfusion-related aerobic muscle metabolism, which manifestation is exercise specific. The training-invariant ACE I-allele-associated differences in heart rate and blood glucose concentration emphasize that the repeated impact of the interval stimulus, despite a near doubling of the initial metabolic load, was insufficient to overturn ACE-related genetic influences on cardiovascular function.

Association of Gene Variants with Seasonal Variation in Muscle Strength and Aerobic Capacity in Elite Skiers.

Background: The training of elite skiers follows a systematic seasonal periodization with a preparation period, when anaerobic muscle strength, aerobic capacity, and cardio-metabolic recovery are specifically conditioned to provide extra capacity for developing ski-specific physical fitness in the subsequent competition period. We hypothesized that periodization-induced alterations in muscle and metabolic performance demonstrate important variability, which in part is explained by gene-associated factors in association with sex and age. Methods: A total of 34 elite skiers (20.4 ± 3.1 years, 19 women, 15 men) underwent exhaustive cardiopulmonary exercise and isokinetic strength testing before and after the preparation and subsequent competition periods of the World Cup skiing seasons 2015-2018. Biometric data were recorded, and frequent polymorphisms in five fitness genes, ACE-I/D (rs1799752), TNC (rs2104772), ACTN3 (rs1815739), and PTK2 (rs7460, rs7843014), were determined with specific PCR reactions on collected DNA. Relative percentage changes of cardio-pulmonary and skeletal muscle metabolism and performance over the two seasonal periods were calculated for 160 data points and subjected to analysis of variance (ANOVA) to identify hypothesized and novel associations between performance alterations and the five respective genotypes and determine the influence of age × sex. A threshold of 0.1 for the effect size (h2) was deemed appropriate to identify relevant associations and motivate a post hoc test to localize effects. Results: The preparation and competition periods produced antidromic functional changes, the extent of which varied with increasing importance for anaerobic strength, aerobic performance, cardio-metabolic efficiency, and cardio-metabolic/muscle recovery. Only peak RER (-14%), but not anaerobic strength and peak aerobic performance, and parameters characterizing cardio-metabolic efficiency, differed between the first and last studied skiing seasons because improvements over the preparation period were mostly lost over the competition period. A number of functional parameters demonstrated associations of variability in periodic changes with a given genotype, and this was considerably influenced by athlete "age", but not "sex". This concerned age-dependent associations between periodic changes in muscle-related parameters, such as anaerobic strength for low and high angular velocities of extension and flexion and blood lactate concentration, with rs1799752 and rs2104772, whose gene products relate to sarcopenia. By contrast, the variance in period-dependent changes in body mass and peak VO2 with rs1799752 and rs2104772, respectively, was independent of age. Likely, the variance in periodic changes in the reliance of aerobic performance on lactate, oxygen uptake, and heart rate was associated with rs1815739 independent of age. These associations manifested at the post hoc level in genotype-associated differences in critical performance parameters. ACTN3 T-allele carriers demonstrated, compared to non-carriers, largely different periodic changes in the muscle-associated parameters of aerobic metabolism during exhaustive exercise, including blood lactate and respiration exchange ratio. The homozygous T-allele carriers of rs2104772 demonstrated the largest changes in extension strength at low angular velocity during the preparation period. Conclusions: Physiological characteristics of performance in skiing athletes undergo training period-dependent seasonal alterations the extent of which is largest for muscle metabolism-related parameters. Genotype associations for the variability in changes of aerobic metabolism-associated power output during exhaustive exercise and anaerobic peak power over the preparation and competition period motivate personalized training regimes. This may help to predict and maximize the benefit of physical conditioning of elite skiers based on chronological characteristics and the polymorphisms of the ACTN3, ACE, and TNC genes investigated here.