Tenomodulin is Required for Tendon Endurance Running and Collagen I Fibril Adaptation to Mechanical Load.

Tendons are dense connective tissues that attach muscles to bone with an indispensable role in locomotion because of their intrinsic properties of storing and releasing muscle- generated elastic energy. Tenomodulin (Tnmd) is a well-accepted gene marker for the mature tendon/ligament lineage and its loss-of -function in mice leads to a phenotype with distinct signs of premature aging on tissue and stem/progenitor cell levels. Based on these findings, we hypothesized that Tnmd might be an important factor in the functional performance of tendons. Firstly, we revealed that Tnmd is a mechanosensitive gene and that the C-terminus of the protein co-localize with collagen I-type fibers in the extracellular matrix. Secondly, using an endurance training protocol, we compared Tnmd knockout mice with wild types and showed that Tnmd deficiency leads to significantly inferior running performance that further worsens with training. In these mice, endurance running was hindered due to abnormal response of collagen I cross-linking and proteoglycan genes leading to an inadequate collagen I fiber thickness and elasticity. In sum, our study demonstrates that Tnmd is required for proper tendon tissue adaptation to endurance running and aids in better understanding of the structural-functional relationships of tendon tissues.

p38 MAPK activation and H3K4 trimethylation is decreased by lactate in vitro and high intensity resistance training in human skeletal muscle.

Exercise induces adaptation of skeletal muscle by acutely modulating intracellular signaling, gene expression, protein turnover and myogenic activation of skeletal muscle stem cells (Satellite cells, SCs). Lactate (La)-induced metabolic stimulation alone has been shown to modify SC proliferation and differentiation. Although the mechanistic basis remains elusive, it was demonstrated that La affects signaling via p38 mitogen activated protein kinase (p38 MAPK) which might contribute to trimethylation of histone 3 lysine 4 (H3K4me3) known to regulate satellite cell proliferation and differentiation. We investigated the effects of La on p38 MAPK and H3K4me3 in a model of activated SCs. Differentiating C2C12 myoblasts were treated with La (20 mM) and samples analysed using qRT-PCR, immunofluorescence, and western blotting. We determined a reduction of p38 MAPK phosphorylation, decreased H3K4me3 and reduced expression of Myf5, myogenin, and myosin heavy chain (MHC) leading to decreased differentiation of La-treated C2C12 cells after 5 days of repeated La treatment. We further investigated whether this regulatory pathway would be affected in human skeletal muscle by the application of two different resistance exercise regimes (RE) associated with distinct metabolic demands and blood La accumulation. Muscle biopsies were obtained 15, 30 min, 1, 4, and 24 h post exercise after moderate intensity RE (STD) vs. high intensity RE (HIT). Consistent with in vitro results, reduced p38 phosphorylation and blunted H3K4me3 were also observed upon metabolically demanding HIT RE in human skeletal muscle. Our data provide evidence that La-accumulation acutely affects p38 MAPK signaling, gene expression and thereby cell differentiation and adaptation in vitro, and likely in vivo.

Dropped head congenital muscular dystrophy caused by de novo mutations in LMNA.

BACKGROUND: Dropped head syndrome is an easily recognizable clinical presentation of Lamin A/C-related congenital muscular dystrophy. Patients usually present in the first year of life with profound neck muscle weakness, dropped head, and elevated serum creatine kinase. CASE DESCRIPTION: Two patients exhibited head drop during infancy although they were able to sit independently. Later they developed progressive axial and limb-girdle weakness. Creatine kinase levels were elevated and muscle biopsies of both patients showed severe dystrophic changes. The distinctive clinical hallmark of the dropped head led us to the diagnosis of Lamin A/C-related congenital muscular dystrophy, with a pathogenic de novo mutation p.Glu31del in the head domain of the Lamin A/C gene in both patients. Remarkably, one patient also had a central involvement with white matter changes on brain magnetic resonance imaging. CONCLUSION: Lamin A/C-related dropped-head syndrome is a rapidly progressive congenital muscular dystrophy and may lead to loss of ambulation, respiratory insufficiency, and cardiac complications. Thus, the genetic diagnosis of dropped-head syndrome as L-CMD and the implicated clinical care protocols are of vital importance for these patients. This disease may be underdiagnosed, as only a few genetically confirmed cases have been reported.

Intense Resistance Exercise Promotes the Acute and Transient Nuclear Translocation of Small Ubiquitin-Related Modifier (SUMO)-1 in Human Myofibres.

Protein sumoylation is a posttranslational modification triggered by cellular stress. Because general information concerning the role of small ubiquitin-related modifier (SUMO) proteins in adult skeletal muscle is sparse, we investigated whether SUMO-1 proteins will be subjected to time-dependent changes in their subcellular localization in sarcoplasmic and nuclear compartments of human type I and II skeletal muscle fibers in response to acute stimulation by resistance exercise (RE). Skeletal muscle biopsies were taken at baseline (PRE), 15, 30, 60, 240 min and 24 h post RE from 6 male subjects subjected to a single bout of one-legged knee extensions. SUMO-1 localization was determined via immunohistochemistry and confocal laser microscopy. At baseline SUMO-1 was localized in perinuclear regions of myonuclei. Within 15 and up to 60 min post exercise, nuclear SUMO-1 localization was significantly increased (p < 0.01), declining towards baseline levels within 240 min post exercise. Sarcoplasmic SUMO-1 localization was increased at 15 min post exercise in type I and up to 30 min post RE in type II myofibres. The changing localization of SUMO-1 proteins acutely after intense muscle contractions points to a role for SUMO proteins in the acute regulation of the skeletal muscle proteome after exercise.

Homozygous mutation in Atlastin GTPase 1 causes recessive hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is an extremely heterogeneous disease caused by mutations of numerous genes leading to lower limb spasticity (pure forms) that can be accompanied by neurological symptoms (complex forms). Despite recent advances, many causal mutations in patients remain unknown. We identified a consanguineous family with the early-onset HSP. Whole-exome sequencing revealed homozygosity for a novel Atlastin GTPase 1 gene stop mutation in three affected siblings. Heterozygous parents and siblings were unaffected. This was unexpected as mutations in the Atlastin 1 gene are known to cause autosomal dominant HSP. But our study showed that Atlastin 1 mutations may cause autosomal recessively inherited paraplegia with an underlying loss-of-function mechanism. Hence, patients with recessive forms of HSP should also be tested for the Atlastin 1 gene.

State of the art in cell-cell fusion.

Mammalian life begins with a cell-cell fusion event, i.e. the fusion of the spermatozoid with the oocyte and needs further cell-cell fusion processes for the development, growth, and maintenance of tissues and organs over the whole life span. Furthermore, cellular fusion plays a role in infection, cancer, and stem cell-dependent regeneration as well as including an expanded meaning of partial cellular fusion, nanotube formation, and microparticle-cell fusion. The cellular fusion process is highly regulated by proteins which carry the information to organize and regulate membranes allowing the merge of two separate lipid bilayers into one. The regulation of this genetically and epigenetically controlled process is achieved by different kinds of signals leading to communication of fusing cells. The local cellular and extracellular environment additionally initiates specific cell signaling necessary for the induction of the cell-cell fusion process. Common motifs exist in distinct cell-cell fusion processes and their regulation. However, there is specific regulation of different cell-cell fusion processes, e.g. myoblast, placental, osteoclast, and stem cell fusion. Hence, specialized fusion events vary between cell types and species. Molecular mechanisms remain largely unknown, especially limited knowledge is present for cancer and stem cell fusion mechanisms and regulation. More research is necessary for the understanding of cellular fusion processes which can lead to development of new therapeutic strategies grounding on cellular fusion regulation.

Stimulation of soluble guanylyl cyclase protects against obesity by recruiting brown adipose tissue.

Obesity is characterized by a positive energy balance and expansion of white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) combusts energy to produce heat. Here we show that a small molecule stimulator (BAY 41-8543) of soluble guanylyl cyclase (sGC), which produces the second messenger cyclic GMP (cGMP), protects against diet-induced weight gain, induces weight loss in established obesity, and also improves the diabetic phenotype. Mechanistically, the haeme-dependent sGC stimulator BAY 41-8543 enhances lipid uptake into BAT and increases whole-body energy expenditure, whereas ablation of the haeme-containing ß1-subunit of sGC severely impairs BAT function. Notably, the sGC stimulator enhances differentiation of human brown adipocytes as well as induces 'browning' of primary white adipocytes. Taken together, our data suggest that sGC is a potential pharmacological target for the treatment of obesity and its comorbidities.

High force development augments skeletal muscle signalling in resistance exercise modes equalized for time under tension.

How force development and time under tension (TUT) during resistance exercise (RE) influence anabolic signalling of skeletal muscle is incompletely understood. We hypothesized that high force development during RE is more important for post-exercise-induced signalling than submaximal and fatiguing RE with lower force development but similar TUT. Twenty-two male subjects (24 ± 6 years, 181 ± 9 cm, 79 ± 2 kg) performed three distinct RE modes in the fed state with equal TUT but distinct force output: (i) maximal eccentric RE (ECC, n = 7) three sets, eight reps, 100% eccentric dynamic force; (ii) standard RE (STD, n = 7), three sets, 10 reps, 75% dynamic force; and (iii) high fatiguing single-set RE (HIT, n = 8), 20 reps, 100% eccentric-concentric force; vastus lateralis biopsies were collected at baseline, 15, 30, 60, 240 min and 24 h after RE, and the signalling of mechanosensitive and mammalian target of rapamycin (mTOR)-related proteins was determined. The phosphorylation levels of pFAK(Tyr397), pJNK(Thr183/Tyr185), pAKT(Thr308/Ser473), pmTOR(Ser2448), p4E-BP1(Thr37/46), p70s6k(Thr389)/(Ser421/Thr424) and pS6(Ser235/236) were significantly higher in ECC than those in STD and HIT at several time points (P < 0.01). pJNK(Thr183/Tyr185) and pS6(Ser235/236) levels were significantly higher in type II myofibres in ECC compared with STD and HIT. HIT exerted throughout the weakest signalling response. We conclude that high force development during acute RE is superior for anabolic skeletal muscle signalling than fatiguing RE with lower force output but similar TUT. Our results suggest that this response is substantially driven by the higher activation of type II myofibres during RE.

Lactate regulates myogenesis in C2C12 myoblasts in vitro.

Satellite cells (SCs) are the resident stem cells of skeletal muscle tissue which play a major role in muscle adaptation, e.g. as a response to physical training. The aim of this study was to examine the effects of an intermittent lactate (La) treatment on the proliferation and differentiation of C2C12 myoblasts, simulating a microcycle of high intensity endurance training. Furthermore, the involvement of reactive oxygen species (ROS) in this context was examined. C2C12 myoblasts were therefore repeatedly incubated for 2 h each day with 10 mM or 20 mM La differentiation medium (DM) and in some cases 20 mM La DM plus different antioxidative substances for up to 5 days. La free (0 mM) DM served as a control. Immunocytochemical staining, Western blot analysis and colorimetric assays were used to assess oxidative stress, proliferation, and differentiation. Results show that La induces oxidative stress, enhances cell-cycle withdrawal, and initiates early differentiation but delays late differentiation in a timely and dose-dependent manner. These effects can be reversed by the addition of antioxidants to the La DM. We therefore conclude that La has a regulatory role in C2C12 myogenesis via a ROS-sensitive mechanism which elicits implications for reassessing some aspects of training and the use of nutritional supplements.

Intense resistance exercise induces early and transient increases in ryanodine receptor 1 phosphorylation in human skeletal muscle.

BACKGROUND: While ryanodine receptor 1 (RyR1) critically contributes to skeletal muscle contraction abilities by mediating Ca²âºion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK) senses contraction-induced energetic stress by phosphorylation at Thr¹7². Phosphorylation of RyR1 at serine²84³ (pRyR1Ser²84³) results in leaky RyR1 channels and impaired Ca²âºhomeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²âº-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. PURPOSE: Determine the effects and early time-course of resistance exercise on pRyR1Ser²84³ and pAMPKThr¹7² in type I and II myofibers. METHODS: 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg) performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²84³ and pAMPKThr¹7² levels were determined by western blot and semi-quantitative immunohistochemistry techniques. RESULTS: While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²84³ increased 15 min and peaked 30 min (p<0.01) post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²84³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05). pAMPKThr¹7² also increased 15 to 30 min post exercise (p<0.01) in type I and II myofibers and in whole skeletal muscle. CONCLUSION: Resistance exercise induces acutely increased pRyR1Ser²84³ and concomitantly pAMPKThr¹7² levels for up to 30 min in resistance exercised myofibers. This provides a time-course by which pRyR1Ser²84³ can mechanistically impact Ca²âºhandling properties and consequently induce reduced myofiber contractility beyond immediate fatiguing mechanisms.

Food and macronutrient intake of elite Ethiopian distance runners.

BACKGROUND: Explanations for the phenomenal success of East African distance runners include unique dietary practices. The aim of the present study was to assess the food and macronutrient intake of elite Ethiopian distance runners during a period of high intensity exercise training at altitude and prior to major competition. METHODS: The dietary intake of 10 highly-trained Ethiopian long distance runners, living and training at high altitude (approximately 2400 m above sea level) was assessed during a 7 day period of intense training prior to competition using the standard weighed intake method. Training was also assessed using an activity/training diary. RESULTS: Body mass was stable (i.e., was well maintained) over the assessment period (pre: 56.7 ± 4.3 kg vs. post: 56.6 ± 4.2 kg, P = 0.54; mean ± SD). The diet comprised of 13375 ± 1378 kJ and was high in carbohydrate (64.3 ± 2.6%, 545 ± 49 g, 9.7 ± 0.9 g/kg). Fat and protein intake was 23.3 ± 2.1% (83 ± 14 g) and 12.4 ± 0.6% (99 ± 13 g, 1.8 ± 0.2 g/kg), respectively. Fluid intake comprised mainly of water (1751 ± 583 mL), while no fluids were consumed before or during training with only modest amounts being consumed following training. CONCLUSIONS: Similar to previous studies in elite Kenyan distance runners, the diet of these elite Ethiopian distance runners met most recommendations of endurance athletes for macronutrient intake but not for fluid intake.