Growth hormone/IGF-I-dependent signaling restores decreased expression of the myokine SPARC in aged skeletal muscle.

Skeletal muscle exerts many beneficial effects on the human body including the contraction-dependent secretion of peptides termed myokines. We have recently connected the myokine secreted protein acidic and rich in cysteine (SPARC) to the formation of intramuscular adipose tissue (IMAT) in skeletal muscle from aged mice and humans. Here, we searched for inducers of SPARC in order to uncover novel treatment approaches for IMAT. Endurance exercise in mice as well as forskolin treatment in vitro only modestly activated SPARC levels. However, through pharmacological treatments in vitro, we identified IGF-I as a potent inducer of SPARC expression in muscle cells, likely through a direct activation of its promoter via phosphatidylinositol 4,5-bisphospate 3-kinase (PI3K)-dependent signaling. We employed two different mouse models of growth hormone (GH)/IGF-I deficiency to solidify our understanding of the relationship between IGF-I and SPARC in vivo. GH administration robustly increased intramuscular SPARC levels (3.5-fold) in GH releasing hormone receptor-deficient mice and restored low intramuscular SPARC expression in skeletal muscle from aged mice. Intramuscular glycerol injections induced higher levels of adipocyte markers (adiponectin, perilipin) in aged compared to young mice, which was not prevented by GH treatment. Our study provides a roadmap for the study of myokine regulation during aging and demonstrates that the GH/IGF-I axis is critical for SPARC expression in skeletal muscle. Although GH treatment did not prevent IMAT formation in the glycerol model, targeting SPARC by exercise or by activation of IGF-I signaling might offer a novel therapeutic strategy against IMAT formation during aging. KEY MESSAGES : IGF-I regulates the myokine SPARC in muscle cells directly at the promoter level. GH/IGF-I is able to restore the decreased SPARC levels in aged skeletal muscle. The glycerol model induces higher adipocyte markers in aged compared to young muscle. GH treatment does not prevent IMAT formation in the glycerol model.

FGF-2-dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis.

Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2-dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle.

Genetic deletion of microRNA biogenesis in muscle cells reveals a hierarchical non-clustered network that controls focal adhesion signaling during muscle regeneration.

OBJECTIVE: Decreased muscle mass is a major contributor to age-related morbidity, and strategies to improve muscle regeneration during ageing are urgently needed. Our aim was to identify the subset of relevant microRNAs (miRNAs) that partake in critical aspects of muscle cell differentiation, irrespective of computational predictions, genomic clustering or differential expression of the miRNAs. METHODS: miRNA biogenesis was deleted in primary myoblasts using a tamoxifen-inducible CreLox system and combined with an add-back miRNA library screen. RNA-seq experiments, cellular signalling events, and glycogen synthesis, along with miRNA inhibitors, were performed in human primary myoblasts. Muscle regeneration in young and aged mice was assessed using the cardiotoxin (CTX) model. RESULTS: We identified a hierarchical non-clustered miRNA network consisting of highly (miR-29a), moderately (let-7) and mildly active (miR-125b, miR-199a, miR-221) miRNAs that cooperate by directly targeting members of the focal adhesion complex. Through RNA-seq experiments comparing single versus combinatorial inhibition of the miRNAs, we uncovered a fundamental feature of this network, that miRNA activity inversely correlates to miRNA cooperativity. During myoblast differentiation, combinatorial inhibition of the five miRNAs increased activation of focal adhesion kinase (FAK), AKT, and p38 mitogen-activated protein kinase (MAPK), and improved myotube formation and insulin-dependent glycogen synthesis. Moreover, antagonizing the miRNA network in vivo following CTX-induced muscle regeneration enhanced muscle mass and myofiber formation in young and aged mice. CONCLUSION: Our results provide novel insights into the dynamics of miRNA cooperativity and identify a miRNA network as therapeutic target for impaired focal adhesion signalling and muscle regeneration during ageing.

Acute Impact of Recovery on the Restoration of Cellular Immunological Homeostasis.

In view of the growing amount of (intense) training in competitive sports, quick recovery plays a superior role in performance restoration. The aim of the present study was to compare the effects of active versus passive recovery during high-intensity interval training (HIIT) and sprint interval training (SIT) protocols on acute alterations of circulating blood cells. Twelve male triathletes/cyclists performed 1) a HIIT consisting of 4×4 min intervals, 2) a SIT consisting of 4×30s intervals, separated by either active or passive recovery. Blood samples were collected immediately before and at 0', 30', 60' and 180' (minutes) post-exercise. Outcomes comprised leukocytes, lymphocytes, neutrophils, mixed cell count, platelets, cellular inflammation markers (neutrophil/lymphocyte-ratio (NLR), platelet/lymphocyte-ratio (PLR)), and the systemic immune-inflammation index (SII). In view of HIIT, passive recovery attenuated the changes in lymphocytes and neutrophils compared to active recovery. In view of SIT, active recovery attenuated the increase in leukocytes, lymphocytes and absolute mixed cell count compared to passive recovery. Both protocols, independent of recovery, significantly increased NLR, PLR and SII up to 3h of recovery compared to pre-exercise values. The mode of recovery influences short-term alterations in the circulating fraction of leukocytes, lymphocytes, neutrophils and the mixed cell count, which might be associated with different hormonal and metabolic stress responses due to the mode of recovery.

Evidence for skeletal muscle fiber type-specific expressions of mechanosensors.

Mechanosensors govern muscle tissue integrity and constitute a subcellular structure known as costameres. Costameres physically link the muscle extracellular matrix to contractile and signaling 'hubs' inside muscle fibers mainly via integrins and are localized beneath sarcolemmas of muscle fibers. Costameres are the main mechanosensors converting mechanical cues into biological events. However, the fiber type-specific costamere architecture in muscles is unexplored. We hypothesized that fiber types differ in the expression of genes coding for costamere components. By coupling laser microdissection to a multiplex tandem qPCR approach, we demonstrate that type 1 and type 2 fibers indeed show substantial differences in their mechanosensor complexes. We confirmed these data by fiber type population-specific protein analysis and confocal microscopy-based localization studies. We further show that knockdown of the costamere gene integrin-linked kinase (Ilk) in muscle precursor cells results in significantly increased slow-myosin-coding Myh7 gene, while the fast-myosin-coding genes Myh1, Myh2, and Myh4 are downregulated. In parallel, protein synthesis-enhancing signaling molecules (p-mTORSer2448, p < 0.05; p-P70S6KThr389, tendency with p < 0.1) were reduced upon Ilk knockdown. However, overexpression of slow type-inducing NFATc1 in muscle precursor cells did not change Ilk or other costamere gene expressions. In addition, we demonstrate fiber type-specific costamere gene regulation upon mechanical loading and unloading conditions. Our data imply that costamere genes, such as Ilk, are involved in the control of muscle fiber characteristics. Further, they identify costameres as muscle fiber type-specific loading management 'hubs' and may explain adaptation differences of muscle fiber types to mechanical (un)loading.

Uncoupling protein 1 expression in adipocytes derived from skeletal muscle fibro/adipogenic progenitors is under genetic and hormonal control.

BACKGROUND: Intramuscular fatty infiltration is generally associated with the accumulation of white adipocytes in skeletal muscle and unfavourable metabolic outcomes. It is, however, still unclear whether intramuscular adipocytes could also acquire a brown-like phenotype. Here, we detected intramuscular expression of brown adipocyte markers during fatty infiltration in an obesity-resistant mouse strain and extensively compared the potential of two different stem cell populations residing in skeletal muscle to differentiate into brown-like adipocytes. METHODS: Fatty infiltration was induced using intramuscular glycerol or cardiotoxin injection in the tibialis anterior muscles of young or aged 129S6/SvEvTac (Sv/129) mice or interleukin-6 (IL-6) knockout mice, and the expression of general and brown adipocyte markers was assessed after 4 weeks. Fibro/adipogenic progenitors (FAPs) and myogenic progenitors were prospectively isolated using fluorescence-activated cell sorting from skeletal muscle of male and female C57Bl6/6J and Sv/129 mice, and monoclonal and polyclonal cultures were treated with brown adipogenic medium. Additionally, FAPs were differentiated with medium supplemented or not with triiodothyronine. RESULTS: Although skeletal muscle expression of uncoupling protein 1 (Ucp1) was barely detectable in uninjected tibialis anterior muscle, it was drastically induced following intramuscular adipogenesis in Sv/129 mice and further increased in response to beta 3-adrenergic stimulation. Intramuscular Ucp1 expression did not depend on IL-6 and was preserved in aged skeletal muscle. Myogenic progenitors did not form adipocytes neither in polyclonal nor monoclonal cultures. Fibro/adipogenic progenitors, on the other hand, readily differentiated into brown-like, UCP1+ adipocytes. Uncoupling protein 1 expression in differentiated FAPs was regulated by genetic background, sex, and triiodothyronine treatment independently of adipogenic differentiation levels. CONCLUSIONS: Intramuscular adipogenesis is associated with increased Ucp1 expression in skeletal muscle from obesity-resistant mice. Fibro/adipogenic progenitors provide a likely source for intramuscular adipocytes expressing UCP1 under control of both genetic and hormonal factors. Therefore, FAPs constitute a possible target for therapies aiming at the browning of intramuscular adipose tissue and the metabolic improvement of skeletal muscle affected by fatty infiltration.

Reply to: Could superimposed electromyostimulation be an effective training to improve aerobic and anaerobic capacity? Methodological considerations for its development.

In the present reply, we would like to put forward our perspective on the points raised by Amaro-Gahete et al. The main concerns addressed by the authors deal with the stimulation modalities applied in our study, compared to the results of Miyamoto et al. (Eur J Sports Sci 16(8):1104-1110, 2016), who showed an increase in the first ventilatory threshold and VO2peak after 16 × 30 min low-frequency neuromuscular electrical stimulation (NMES). We have to emphasize that both mentioned studies generally follow different approaches, and that the different outcomes might not only be related to stimulation modalities. Even the results of different studies, which used sole NMES is not consistent. Especially the relevance of local metabolic and ultrastructural adaptations in skeletal muscle for the translation to functional performance, that is particularly important for sport and activities, is not always investigated in these studies.

Chronic effects of superimposed electromyostimulation during cycling on aerobic and anaerobic capacity.

PURPOSE: To examine if chronic endurance training by means of simultaneously applied, superimposed electromyostimulation (EMS) can be used to improve performance and physiological core parameters compared to the traditional cycling. METHODS: Twenty-one male subjects (VO2peak 55.2 ± 5.1 ml min- 1 kg- 1) were assigned to either a cycling (C) or cycling with superimposed EMS (C + E) group. Before and after the 4-week training period, including 14 sessions of moderate cycling [60 min at 60% peak power output (PPO)], participants performed a 20-min time-trial, a step test to exhaustion, a 30-s isokinetic sprint test, and maximum force- and power-tests. Markers of muscle damage and metabolic condition were assessed during the training period. RESULTS: Step test results revealed increases in PPO, VO2peak, lactate threshold 1, and the anaerobic threshold for both groups (p < 0.05). Mean power output (MPO) obtained from time-trial was improved in C and C + E (p < 0.05). Isokinetic sprint test revealed increased PPO in both groups, whereas MPO was only changed in C (p < 0.05). Strength parameters were unaffected. Although metabolic stimuli and markers of muscle damage were higher in C + E compared to C, improvements of endurance performance and capacity were not significantly different between C and C + E. CONCLUSIONS: Despite a higher metabolic, respiratory, and muscular demand, chronic additional superimposed EMS during cycling does not result in superior improvements in endurance and strength performance compared to the traditional cycling.

Impact of high-intensity and high-volume exercise on short-term perturbations in the circulating fraction of different cell types.

BACKGROUND: High-intensity training (HIT) can improve endurance performance and VO2max more effectively than high-volume training (HVT). Hence, the implementation of HIT protocols such as 4x30 s "all-out" and 4x4 min at 90-95% VO2max is currently existent in various sports. However, there is limited awareness of the acute changes in blood cell count following these protocols. Therefore, the purpose of the study was to examine the acute effects on circulating leukocyte differential count (LDC) by comparing the two HIT interventions with a single HVT intervention. METHODS: Twelve healthy triathletes/cyclists (VO2peak: 64.3±9.7 mL·kg-1·min-1) participated in the study. Subjects performed: 1) a two-hours low-intensity exercise at an intensity of 55% peak power output (PPO); 2) 4x4 min interval bouts at an intensity of 90-95% PPO; 3) 4x30 s "all-out". Blood samples were taken immediately before exercise (pre) and 0', 30', 60' and 180' postexercise. Biphasic leukocyte enumeration was different between both HIT protocols and nonexistent after high-volume exercise. RESULTS: Data revealed significant time and intervention effects for leukocytes, lymphocytes and neutrophils. After 4x30 s lymphocytes were significantly higher 0´ postintervention compared to 4x4 min and high-volume exercise. Furthermore, concentrations of leukocytes and neutrophils were significantly higher after the "all-out" protocol compared to 4x4 min at 180' postexercise. CONCLUSIONS: The results suggest that 4x30 s result in larger short-term perturbations in the circulating fraction of leukocytes compared to 4x4 min, which might be associated with increased hormonal and metabolic stress responses after 4x30 s.

Effects of active vs. passive recovery during Wingate-based training on the acute hormonal, metabolic and psychological response.

OBJECTIVE: The exercise-induced metabolic stress can be influenced by the mode of recovery and is associated with acute hormonal responses. Therefore, it is hypothesized that active recovery between high intensity intervals reduces the metabolic stimulus and therefore the hormonal response compared to passive recovery. DESIGN: 12 male cyclist/triathletes performed four 30s all-out intervals, either with active (A) or passive (P) recovery between each bout. Human growth hormone (hGH), testosterone and cortisol, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and macrophage migration inhibitory factor (MIF) were determined pre, 0', 30', 60' and 180' after both interventions. Metabolic stimuli and perturbations were characterized by lactate, blood gas (pH, BE, HCO3(-), PO2, PCO2), and spirometric analysis. RESULTS: Both interventions caused a transient increase in circulating levels of cortisol, testosterone, testosterone/cortisol-ratio, hGH, VEGF and HGF. Transient differences between A- and P-recovery were found only for testosterone and HGF directly after exercise, despite significant differences in metabolic disturbances (lactate, acid base status). CONCLUSION: Based on the data of testosterone, hGH and the testosterone/cortisol-ratio, as well as on the data of VEGF and HGF it appears that this kind of exercise protocol may promote anabolic processes and may lead to pro-angiogenic conditions independent of the mode of recovery. However transient differences between A- and P-recovery were shown for testosterone and HGF. In contrast, cortisol and hGH, which are known to be sensitive for metabolic perturbations (e.g. pH) showed no differences. Therefore, it is proposed that if a certain threshold for metabolic perturbations is exceeded, a hormonal response is induced, which does not differ between A- and P-recovery.