Widespread Vascularization and Correlation of Glycosaminoglycan Accumulation to Tendon Pain in Human Plantar Fascia Tendinopathy.

BACKGROUND: Plantar fasciitis is a painful tendinous condition (tendinopathy) with a high prevalence in athletes. While a healthy tendon has limited blood flow, ultrasound has indicated elevated blood flow in tendinopathy, but it is unknown if this is related to a de facto increase in the tendon vasculature. Likewise, an accumulation of glycosaminoglycans (GAGs) is observed in tendinopathy, but its relationship to clinical pain is unknown. PURPOSE: To explore to what extent vascularization, inflammation, and fat infiltration were present in patients with plantar fasciitis and if they were related to clinical symptoms. STUDY DESIGN: Descriptive laboratory study. METHODS: Biopsy specimens from tendinopathic plantar fascia tissue were obtained per-operatively from both the primary site of tendon pain and tissue swelling ("proximal") and a region that appeared macroscopically healthy at 1 to 2 cm away from the primary site ("distal") in 22 patients. Biopsy specimens were examined with immunofluorescence for markers of blood vessels, tissue cell density, fat infiltration, and macrophage level. In addition, pain during the first step in the morning (registered during an earlier study) was correlated with the content of collagen and GAGs in tissue. RESULTS: High vascularization (and cellularity) was present in both the proximal (0.89%) and the distal (0.96%) plantar fascia samples, whereas inconsistent but not significantly different fat infiltration and macrophage levels were observed. The collagen content was similar in the 2 plantar fascia regions, whereas the GAG content was higher in the proximal region (3.2% in proximal and 2.8% in distal; P = .027). The GAG content in the proximal region was positively correlated with the subjective morning pain score in the patients with tendinopathy (n = 17). CONCLUSION: In patients with plantar fasciitis, marked tissue vascularization was present in both the painful focal region and a neighboring nonsymptomatic area. In contrast, the accumulation of hydrophilic GAGs was greater in the symptomatic region and was positively correlated with increased clinical pain levels in daily life. CLINICAL RELEVANCE: The accumulation of GAGs in tissue rather than the extent of vascularization appears to be linked with the clinical degree of pain symptoms of the disease.

Mechanical properties, collagen and glycosaminoglycan content of equine superficial digital flexor tendons are not affected by training.

Physical activity can activate extracellular matrix (ECM) protein synthesis and influence the size and mechanical properties of tendon. In this study, we aimed to investigate whether different training histories of horses would influence the synthesis of collagen and other matrix proteins and alter the mechanical properties of tendon. Samples from superficial digital flexor tendon (SDFT) from horses that were either (a) currently race trained (n = 5), (b) previously race trained (n = 5) or (c) untrained (n = 4) were analysed for matrix protein abundance (mass spectrometry), collagen and glycosaminoglycan (GAG) content, ECM gene expression and mechanical properties. It was found that ECM synthesis by tendon fibroblasts in vitro varied depending upon the previous training history. In contrast, fascicle morphology, collagen and GAG content, mechanical properties and ECM gene expression of the tendon did not reveal any significant differences between groups. In conclusion, although we could not identify any direct impact of the physical training history on the mechanical properties or major ECM components of the tendon, it is evident that horse tendon cells are responsive to loading in vivo, and the training background may lead to a modification in the composition of newly synthesised matrix.

TNIK is a conserved regulator of glucose and lipid metabolism in obesity.

Obesity and type 2 diabetes (T2D) are growing health challenges with unmet treatment needs. Traf2- and NCK-interacting protein kinase (TNIK) is a recently identified obesity- and T2D-associated gene with unknown functions. We show that TNIK governs lipid and glucose homeostasis in Drosophila and mice. Loss of the Drosophila ortholog of TNIK, misshapen, altered the metabolite profiles and impaired de novo lipogenesis in high sugar-fed larvae. Tnik knockout mice exhibited hyperlocomotor activity and were protected against diet-induced fat expansion, insulin resistance, and hepatic steatosis. The improved lipid profile of Tnik knockout mice was accompanied by enhanced skeletal muscle and adipose tissue insulin-stimulated glucose uptake and glucose and lipid handling. Using the T2D Knowledge Portal and the UK Biobank, we observed associations of TNIK variants with blood glucose, HbA1c, body mass index, body fat percentage, and feeding behavior. These results define an untapped paradigm of TNIK-controlled glucose and lipid metabolism.

GDF15 increases insulin action in the liver and adipose tissue via a ß-adrenergic receptor-mediated mechanism.

Growth differentiation factor 15 (GDF15) induces weight loss and increases insulin action in obese rodents. Whether and how GDF15 improves insulin action without weight loss is unknown. Obese rats were treated with GDF15 and displayed increased insulin tolerance 5 h later. Lean and obese female and male mice were treated with GDF15 on days 1, 3, and 5 without weight loss and displayed increased insulin sensitivity during a euglycemic hyperinsulinemic clamp on day 6 due to enhanced suppression of endogenous glucose production and increased glucose uptake in WAT and BAT. GDF15 also reduced glucagon levels during clamp independently of the GFRAL receptor. The insulin-sensitizing effect of GDF15 was completely abrogated in GFRAL KO mice and also by treatment with the ß-adrenergic antagonist propranolol and in ß1,ß2-adrenergic receptor KO mice. GDF15 activation of the GFRAL receptor increases ß-adrenergic signaling, in turn, improving insulin action in the liver and white and brown adipose tissue.

Achilles Tendon Tissue Turnover Before and Immediately After an Acute Rupture.

BACKGROUND: An Achilles tendon rupture (ATR) is a frequent injury and results in the activation of tendon cells and collagen expression, but it is unknown to what extent turnover of the tendon matrix is altered before or after a rupture. PURPOSE/HYPOTHESIS: The purpose of this study was to characterize tendon tissue turnover before and immediately after an acute rupture in patients. It was hypothesized that a rupture would result in pronounced collagen synthesis in the early phase (first 2 weeks) after the injury. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: The study included patients (N = 18) eligible for surgery after an ATR. At the time of inclusion, the patients ingested deuterium oxide (2H2O) orally, and on the day of surgery (within 14 days of the injury), they received a 3-hour flood-primed infusion of an 15N-proline tracer. During surgery, the patients had 1 biopsy specimen taken from the ruptured part of the Achilles tendon and 1 that was 3 to 5 cm proximal to the rupture as a control. The biopsy specimens were analyzed for carbon-14 (14C) levels in the tissue to calculate long-term turnover (years), incorporation of 2H-alanine (from 2H2O) into the tissue to calculate the fractional synthesis rate (FSR) of proteins in the short term (days), and incorporation of 15N-proline into the tissue to calculate the acute FSR (hours). RESULTS: Both the rupture and the control samples showed consistently lower levels of 14C compared with the predicted level of 14C in a healthy tendon, which indicated increased tendon turnover in a fraction (48% newly synthesized) of the Achilles tendon already for a prolonged period before the rupture. Over the first days after the rupture, the synthesis rate for collagen was relatively constant, and the average synthesis rate on the day of surgery (2-14 days after the rupture) was 0.025% per hour, irrespective of the length of time after a rupture and the site of sampling (rupture vs control). No differences were found in the FSR between the rupture and control samples in the days after the rupture. CONCLUSION: Higher than normal tissue turnover in the Achilles tendon before a rupture indicated that changes in the tendon tissue preceded the injury. In addition, we observed no increase in tendon collagen tissue turnover in the first 2 weeks after an ATR. This favors the view that an increase in the formation of new tendon collagen is not an immediate phenomenon during the regeneration of ruptured tendons in patients. REGISTRATION: NCT03931486 (ClinicalTrials.gov identifier).

TBC1D4-S711 Controls Skeletal Muscle Insulin Sensitization After Exercise and Contraction.

The ability of insulin to stimulate glucose uptake in skeletal muscle is important for whole-body glycemic control. Insulin-stimulated skeletal muscle glucose uptake is improved in the period after a single bout of exercise, and accumulating evidence suggests that phosphorylation of TBC1D4 by the protein kinase AMPK is the primary mechanism responsible for this phenomenon. To investigate this, we generated a TBC1D4 knock-in mouse model with a serine-to-alanine point mutation at residue 711 that is phosphorylated in response to both insulin and AMPK activation. Female TBC1D4-S711A mice exhibited normal growth and eating behavior as well as intact whole-body glycemic control on chow and high-fat diets. Moreover, muscle contraction increased glucose uptake, glycogen utilization, and AMPK activity similarly in wild-type and TBC1D4-S711A mice. In contrast, improvements in whole-body and muscle insulin sensitivity after exercise and contractions were only evident in wild-type mice and occurred concomitantly with enhanced phosphorylation of TBC1D4-S711. These results provide genetic evidence to support that TBC1D4-S711 serves as a major point of convergence for AMPK- and insulin-induced signaling that mediates the insulin-sensitizing effect of exercise and contractions on skeletal muscle glucose uptake.

Distinct myofibre domains of the human myotendinous junction revealed by single-nucleus RNA sequencing.

The myotendinous junction (MTJ) is a specialized domain of the multinucleated myofibre that is faced with the challenge of maintaining robust cell-matrix contact with the tendon under high mechanical stress and strain. Here, we profiled 24,124 nuclei in semitendinosus muscle-tendon samples from three healthy males by using single-nucleus RNA sequencing (snRNA-seq), alongside spatial transcriptomics, to gain insight into the genes characterizing this specialization in humans. We identified a cluster of MTJ myonuclei represented by 47 enriched transcripts, of which the presence of ABI3BP, ABLIM1, ADAMTSL1, BICD1, CPM, FHOD3, FRAS1 and FREM2 was confirmed at the MTJ at the protein level in immunofluorescence assays. Four distinct subclusters of MTJ myonuclei were apparent, comprising two COL22A1-expressing subclusters and two subclusters lacking COL22A1 expression but with differing fibre type profiles characterized by expression of either MYH7 or MYH1 and/or MYH2. Our findings reveal distinct myonuclei profiles of the human MTJ, which represents a weak link in the musculoskeletal system that is selectively affected in pathological conditions ranging from muscle strains to muscular dystrophies.

Disruption of day-to-night changes in circadian gene expression with chronic tendinopathy.

Overuse injury in tendon tissue (tendinopathy) is a frequent and costly musculoskeletal disorder and represents a major clinical problem with unsolved pathogenesis. Studies in mice have demonstrated that circadian clock-controlled genes are vital for protein homeostasis and important in the development of tendinopathy. We performed RNA sequencing, collagen content and ultrastructural analyses on human tendon biopsies obtained 12 h apart in healthy individuals to establish whether human tendon is a peripheral clock tissue and we performed RNA sequencing on patients with chronic tendinopathy to examine the expression of circadian clock genes in tendinopathic tissues. We found time-dependent expression of 280 RNAs including 11 conserved circadian clock genes in healthy tendons and markedly fewer (23) differential RNAs with chronic tendinopathy. Further, the expression of COL1A1 and COL1A2 was reduced at night but was not circadian rhythmic in synchronised human tenocyte cultures. In conclusion, day-to-night changes in gene expression in healthy human patellar tendons indicate a conserved circadian clock as well as the existence of a night reduction in collagen I expression. KEY POINTS: Tendinopathy is a major clinical problem with unsolved pathogenesis. Previous work in mice has shown that a robust circadian rhythm is required for collagen homeostasis in tendons. The use of circadian medicine in the diagnosis and treatment of tendinopathy has been stifled by the lack of studies on human tissue. Here, we establish that the expression of circadian clock genes in human tendons is time dependent, and now we have data to corroborate that circadian output is reduced in diseased tendon tissues. We consider our findings to be of significance in advancing the use of the tendon circadian clock as a therapeutic target or preclinical biomarker for tendinopathy.

Tensile Loaded Tissue-Engineered Human Tendon Constructs Stimulate Myotube Formation.

Skeletal muscle possesses adaptability to mechanical loading and regenerative potential following muscle injury due to muscle stem cell activity. So far, it is known that muscle stem cell activity is supported by the roles of several interstitial cells within skeletal muscle in response to muscle damage. The adjacent tendon is also exposed to repetitive mechanical loading and possesses plasticity like skeletal muscle. However, the interplay between the skeletal muscle and adjacent tendon tissue has not been fully investigated. In this study, we tested whether factors released by three-dimensional engineered human tendon constructs in response to uniaxial tensile loading can stimulate the proliferation and differentiation of human-derived myogenic cells (myoblasts). Tendon constructs were subjected to repetitive mechanical loading (4% strain at 0.5 Hz for 4 h) and nonrepetitive loading (0% strain at 0 Hz for 4 h), and the conditioned media from mechanically loaded and nonmechanically loaded control constructs were applied to myoblasts. Immunofluorescence analysis revealed both an increase of myotube fusion index (≥5 nuclei within one desmin+ myotube) and the myotube diameter when conditioned medium from mechanically loaded tendon constructs was applied. Myostatin, myosin heavy chain 7, and AXIN2 gene expressions were downregulated in myotubes treated with conditioned medium from mechanically loaded tendon constructs. However, proliferative potential (number of Ki67+ and bromodeoxyuridine+ myoblasts) did not differ between the two groups. These results indicate that tendon fibroblasts enhance myotube formation by mechanical loading-induced factors. Our finding suggests that mechanical loading affects the signaling interplay between skeletal muscle and tendon tissue and is thus important for musculoskeletal tissue development and regeneration in humans. Impact statement The interplay between satellite cells and various types of resident cells within the skeletal muscle for muscle regeneration has been extensively studied. However, even though tendon tissue is located adjacent to skeletal muscle tissue and cells in these tissues are exposed to repetitive mechanical loading together, the interaction between muscle and tendon tissues for muscle regeneration remains to be elucidated. In this study, we report that the conditioned media from engineered human tendon tissues undergoing repetitive tensile mechanical loading enhanced myotube formation. Our in vitro findings extend the fundamental understanding of the crosstalk between adjacent tissues of the muscle-tendon unit.

Gene Expression Linked to Reepithelialization of Human Skin Wounds.

Our understanding of the regulatory processes of reepithelialization during wound healing is incomplete. In an attempt to map the genes involved in epidermal regeneration and differentiation, we measured gene expression in formalin-fixed, paraffin-embedded standardized epidermal wounds induced by the suction-blister technique with associated nonwounded skin using NanoString technology. The transcripts of 139 selected genes involved in clotting, immune response to tissue injury, signaling pathways, cell adhesion and proliferation, extracellular matrix remodeling, zinc transport and keratinocyte differentiation were evaluated. We identified 22 upregulated differentially expressed genes (DEGs) in descending order of fold change (MMP1, MMP3, IL6, CXCL8, SERPINE1, IL1B, PTGS2, HBEGF, CXCL5, CXCL2, TIMP1, CYR61, CXCL1, MMP12, MMP9, HGF, CTGF, ITGB3, MT2A, FGF7, COL4A1 and PLAUR). The expression of the most upregulated gene, MMP1, correlated strongly with MMP3 followed by IL6 and IL1B. rhIL-1ß, but not rhIL-6, exposure of cultured normal human epidermal keratinocytes and normal human dermal fibroblasts increased both MMP1 mRNA and MMP-1 protein levels, as well as TIMP1 mRNA levels. The increased TIMP1 in wounds was validated by immunohistochemistry. The six downregulated DEGs (COL7A1, MMP28, SLC39A2, FLG1, KRT10 and FLG2) were associated with epidermal maturation. KLK8 showed the strongest correlation with MKI67 mRNA levels and is a potential biomarker for keratinocyte proliferation. The observed gene expression changes correlate well with the current knowledge of physiological reepithelialization. Thus, the gene expression panel described in this paper could be used in patients with impaired healing to identify possible therapeutic targets.

Human skeletal muscle acetylcholine receptor gene expression in elderly males performing heavy resistance exercise.

Muscle fiber denervation is a major contributor to the decline in muscle mass and function during aging. Heavy resistance exercise is an effective tool for increasing muscle mass and strength, but whether it can rescue denervated muscle fibers remains unclear. Therefore, the purpose of this study was to investigate the potential of heavy resistance exercise to modify indices of denervation in healthy elderly individuals. Thirty-eight healthy elderly men (72 ± 5 yr) underwent 16 wk of heavy resistance exercise, whereas 20 healthy elderly men (72 ± 6 yr) served as nonexercising sedentary controls. Muscle biopsies were obtained pre and post training, and midway at 8 wk. Biopsies were analyzed by immunofluorescence for the prevalence of myofibers expressing embryonic myosin [embryonic myosin heavy chain (MyHCe)], neonatal myosin [neonatal myosin heavy chain (MyHCn)], nestin, and neural cell adhesion molecule (NCAM), and by RT-qPCR for gene expression levels of acetylcholine receptor (AChR) subunits, MyHCn, MyHCe, p16, and Ki67. In addition to increases in strength and type II fiber hypertrophy, heavy resistance exercise training led to a decrease in AChR α1 and ε subunit messenger RNA (mRNA; at 8 wk). Changes in gene expression levels of the α1 and ε AChR subunits with 8 wk of heavy resistance exercise supports the role of this type of exercise in targeting stability of the neuromuscular junction. The number of fibers positive for NCAM, nestin, and MyHCn was not affected, suggesting that a longer timeframe is needed for adaptations to manifest at the protein level.

Human derived tendon cells contribute to myotube formation in vitro.

Skeletal muscle possesses remarkable adaptability to mechanical loading and regenerative potential following muscle injury primarily due to satellite cell activity. Although the roles of several types of interstitial cells in skeletal muscle have been documented, the signaling interplay between the skeletal muscle and the adjacent tendon tissue has not been elucidated. Here, we tested whether human tendon derived cells (tenocytes) could induce human myogenic cells (myoblasts) proliferation and differentiation in vitro using co-culture experiments that allowed us to investigate the effect of tenocytes secretion upon myogenic progression. This was done in vitro by introducing insert wells with either myoblasts, tenocytes, or no cells (control) into a myoblast containing well (co-culture). Immunofluorescence analysis revealed a higher fusion index (≥5 nuclei within one Desmin + myotube) and a higher myotube diameter in co-cultures with tenocytes compared to myoblasts condition. Correspondingly, MHC-IIX gene expression was up-regulated when co-cultured with tenocytes. However, the proliferation of myoblasts (either Ki67 or BrdU + cells) was not enhanced under the presence of tenocytes. These findings show that tenocytes influence myotube formation upon human primary cells in vitro and contribute to understanding the role of tendon derived cells in skeletal muscle during development and regeneration.

Nestin and osteocrin mRNA increases in human semitendinosus myotendinous junction 7 days after a single bout of eccentric exercise.

The myotendinous junction (MTJ), a specialized interface for force transmission between muscle and tendon, has a unique transcriptional activity and is highly susceptible to muscle strain injury. Eccentric exercise training is known to reduce this risk of injury, but knowledge of the influence of exercise on the MTJ at the molecular and cellular levels is limited. In this study, 30 subjects were randomized to a single bout of eccentric exercise 1 week prior to tissue sampling (exercised) or no exercise (control). Samples were collected from the semitendinosus as part of reconstruction of the anterior cruciate ligament and divided into fractions containing muscle, MTJ and tendon, respectively. The concentrations of macrophages and satellite cells were counted, and the expression of genes previously known to be active at the MTJ were analyzed by real-time-quantitative PCR. An effect of the single bout of exercise was found on the expression of nestin (NES) and osteocrin (OSTN) mRNA in the MTJ and tendon fractions. Genes earlier identified at the MTJ (COL22A1, POSTN, ADAMTS8, MNS1, NCAM1) were confirmed to be expressed at a significantly higher level in the MTJ compared to muscle and tendon but were unaffected by exercise. In the exercise group a higher concentration of macrophages, but not of satellite cells, was seen in muscle close to the MTJ. The expression of NES and OSTN was higher in human semitendinosus MTJ 1 week after a single session of heavy eccentric exercise. Based on these results, NES and OSTN could have a part in explaining how the MTJ adapts to eccentric exercise.

Preserved stem cell content and innervation profile of elderly human skeletal muscle with lifelong recreational exercise.

Muscle fibre denervation and declining numbers of muscle stem (satellite) cells are defining characteristics of ageing skeletal muscle. The aim of this study was to investigate the potential for lifelong recreational exercise to offset muscle fibre denervation and compromised satellite cell content and function, both at rest and under challenged conditions. Sixteen elderly lifelong recreational exercisers (LLEX) were studied alongside groups of age-matched sedentary (SED) and young subjects. Lean body mass and maximal voluntary contraction were assessed, and a strength training bout was performed. From muscle biopsies, tissue and primary myogenic cell cultures were analysed by immunofluorescence and RT-qPCR to assess myofibre denervation and satellite cell quantity and function. LLEX demonstrated superior muscle function under challenged conditions. When compared with SED, the muscle of LLEX was found to contain a greater content of satellite cells associated with type II myofibres specifically, along with higher mRNA levels of the beta and gamma acetylcholine receptors (AChR). No difference was observed between LLEX and SED for the proportion of denervated fibres or satellite cell function, as assessed in vitro by myogenic cell differentiation and fusion index assays. When compared with inactive counterparts, the skeletal muscle of lifelong exercisers is characterised by greater fatigue resistance under challenged conditions in vivo, together with a more youthful tissue satellite cell and AChR profile. Our data suggest a little recreational level exercise goes a long way in protecting against the emergence of classic phenotypic traits associated with the aged muscle. KEY POINTS: The detrimental effects of ageing can be partially offset by lifelong self-organized recreational exercise, as evidence by preserved type II myofibre-associated satellite cells, a beneficial muscle innervation status and greater fatigue resistance under challenged conditions. Satellite cell function (in vitro), muscle fibre size and muscle fibre denervation determined by immunofluorescence were not affected by recreational exercise. Individuals that are recreationally active are far more abundant than master athletes, which sharply increases the translational perspective of the present study. Future studies should further investigate recreational activity in relation to muscle health, while also including female participants.

Gene deletion of γ-actin impairs insulin-stimulated skeletal muscle glucose uptake in growing mice but not in mature adult mice.

The cortical cytoskeleton, consisting of the cytoplasmic actin isoforms ß and/or γ-actin, has been implicated in insulin-stimulated GLUT4 translocation and glucose uptake in muscle and adipose cell culture. Furthermore, transgenic inhibition of multiple actin-regulating proteins in muscle inhibits insulin-stimulated muscle glucose uptake. The current study tested if γ-actin was required for insulin-stimulated glucose uptake in mouse skeletal muscle. Based on our previously reported age-dependent phenotype in muscle-specific ß-actin gene deletion (-/- ) mice, we included cohorts of growing 8-14 weeks old and mature 18-32 weeks old muscle-specific γ-actin-/- mice or wild-type littermates. In growing mice, insulin significantly increased the glucose uptake in slow-twitch oxidative soleus and fast-twitch glycolytic EDL muscles from wild-type mice, but not γ-actin-/- . In relative values, the maximal insulin-stimulated glucose uptake was reduced by ~50% in soleus and by ~70% in EDL muscles from growing γ-actin-/- mice compared to growing wild-type mice. In contrast, the insulin-stimulated glucose uptake responses in mature adult γ-actin-/- soleus and EDL muscles were indistinguishable from the responses in wild-type muscles. Mature adult insulin-stimulated phosphorylations on Akt, p70S6K, and ULK1 were not significantly affected by genotype. Hence, insulin-stimulated muscle glucose uptake shows an age-dependent impairment in young growing but not in fully grown γ-actin-/- mice, bearing phenotypic resemblance to ß-actin-/- mice. Overall, γ-actin does not appear required for insulin-stimulated muscle glucose uptake in adulthood. Furthermore, our data emphasize the need to consider the rapid growth of young mice as a potential confounder in transgenic mouse phenotyping studies.

Mutual stimulatory signaling between human myogenic cells and rat cerebellar neurons.

Insight into the bidirectional signaling between primary human myogenic cells and neurons is lacking. For this purpose, human myogenic cells were derived from the semitendinosus and gracilis muscles of five healthy individuals and co-cultured with cerebellar granule neurons from two litters of 7-day-old Wistar rat pups, in muscle medium or neural medium, alongside monocultures of myogenic cells or neurons. RT-PCR was performed to determine human mRNA levels of GAPDH, Ki67, myogenin, and MUSK, and the acetylcholine receptor subtypes CHRNA1, CHRNB1, CHRNG, CHRND, and CHRNE, and rat mRNA levels of GAPDH, Fth1, Rack1, vimentin, Cdh13, and Ppp1r1a. Immunocytochemistry was used to evaluate neurite outgrowth (GAP43) in the presence and absence of myogenic cells. Co-culture with primary neurons lead to higher myogenic cell gene expression levels of GAPDH, myogenin, MUSK, CHRNA1, CHRNG, and CHRND, compared to myogenic cells cultured alone. It appeared that neurons preferentially attached to myotubes and that neurite outgrowth was enhanced when neurons were cultured with myogenic cells compared to monoculture. In neural medium, rat mRNA levels of GAPDH, vimentin, Cdh13, and Ppp1r1a were greater in co-culture, versus monoculture, whereas in muscle medium co-culture lead to lower levels of Fth1, Rack1, vimentin, and Cdh13 than monoculture. These findings demonstrate mutually beneficial stimulatory signaling between rat cerebellar granule neurons and human myogenic cells, providing support for an active role for both the neuron and the muscle cell in stimulating neurite growth and myogenesis. Bidirectional muscle nerve signaling.

Impaired skeletal muscle hypertrophy signaling and amino acid deprivation response in Apoe knockout mice with an unhealthy lipoprotein distribution.

This study explores if unhealthy lipoprotein distribution (LPD) impairs the anabolic and amino acid sensing responses to whey-protein feeding. Thus, if impairment of such anabolic response to protein consumption is seen by the LPD this may negatively affect the skeletal muscle mass. Muscle protein synthesis (MPS) was measured by puromycin labeling in Apolipoprotein E knockout (Apoe KO), characterized by an unhealthy LPD, and wild type mice post-absorptive at 10 and 20 weeks, and post-prandial after whey-protein feeding at 20 weeks. Hypertrophy signaling and amino acid sensing mechanisms were studied and gut microbiome diversity explored. Surprisingly, whey-protein feeding did not affect MPS. p-mTOR and p-4E-BP1 was increased 2 h after whey-protein feeding in both genotypes, but with general lower levels in Apoe KO compared to wild type. At 20 weeks of age, Apoe KO had a greater mRNA-expression for SNAT2, CD98, ATF4 and GCN2 compared to wild type. These responses were not associated with gut microbiota compositional differences. Regardless of LPD status, MPS was similar in Apoe KO and wild type. Surprisingly, whey-protein did not stimulate MPS. However, Apoe KO had lower levels of hypertrophy signaling, was amino acid deprived, and had impaired amino acid sensing mechanisms.

RNA sequencing and immunofluorescence of the myotendinous junction of mature horses and humans.

The myotendinous junction (MTJ) is a specialized interface for transmitting high forces between the muscle and tendon and yet the MTJ is a common site of strain injury with a high recurrence rate. The aim of this study was to identify previously unknown MTJ components in mature animals and humans. Samples were obtained from the superficial digital flexor (SDF) muscle-tendon interface of 20 horses, and the tissue was separated through a sequential cryosectioning approach into muscle, MTJ (muscle tissue enriched in myofiber tips attached to the tendon), and tendon fractions. RT-PCR was performed for genes known to be expressed in the three tissue fractions and t-distributed stochastic neighbor embedding (t-SNE) plots were used to select the muscle, MTJ, and tendon samples from five horses for RNA sequencing. The expression of previously known and unknown genes identified through RNA sequencing was studied by immunofluorescence on human hamstring MTJ tissue. The main finding was that RNA sequencing identified the expression of a panel of 61 genes enriched at the MTJ. Of these, 48 genes were novel for the MTJ and 13 genes had been reported to be associated with the MTJ in earlier studies. The expression of known [COL22A1 (collagen XXII), NCAM (neural cell adhesion molecule), POSTN (periostin), NES (nestin), OSTN (musclin/osteocrin)] and previously undescribed [MNS1 (meiosis-specific nuclear structural protein 1), and LCT (lactase)] MTJ genes was confirmed at the protein level by immunofluorescence on tissue sections of human MTJ. In conclusion, in muscle-tendon interface tissue enriched with myofiber tips, we identified the expression of previously unknown MTJ genes representing diverse biological processes, which may be important in the maintenance of the specialized MTJ.

No Treatment Benefits of Local Administration of Insulin-like Growth Factor-1 in Addition to Heavy Slow Resistance Training in Tendinopathic Human Patellar Tendons: A Randomized, Double-Blind, Placebo-Controlled Trial With 1-Year Follow-up.

BACKGROUND: Heavy slow resistance (HSR) training is currently recommended as part of the treatment of patellar tendon tendinopathy. However, treatment success is not reached in all patients, and combinations of different treatments could be beneficial. Local administration of insulin-like growth factor-1 (IGF-1) in humans has been shown to quickly stimulate tendon collagen synthesis. PURPOSE: To study whether IGF-1 injections combined with HSR training enhance tendon synthesis, tissue structure, and patient satisfaction versus saline injection combined with HSR training in patients with patellar tendinopathy. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: Forty patients (age 18-50 years) with unilateral patellar tendinopathy undertook HSR training (3 times a week for 12 weeks) and received intratendinous IGF-1 injections (1 mg IGF-1 per dose) or isotonic saline injections (sham injections) at baseline and after 1 and 2 weeks of training. The primary outcome was collagen synthesis parameters after 12 weeks (primary endpoint). The secondary outcomes were patient-reported outcomes (scores on the Victorian Institute of Sport Assessment-Patella [VISA-P] and visual analog scale [VAS] for pain) and structural changes before the initiation of treatment and at week 3, week 12, and 1 year after the initiation of treatment. RESULTS: Analysis of the patellar tendon biopsy specimens at 12 weeks showed that collagen mRNA and total RNA were increased in the tendinopathic tendons compared with the contralateral healthy tendons regardless of treatment with IGF-1 or saline. Similarly, no difference between the groups was seen in tendon thickness and Doppler activity at week 12 or at 1-year follow-up. The combination of HSR training and IGF-1 injections significantly improved VISA-P and VAS pain scores after 3 weeks, whereas the overall responses after 12 weeks and at 1-year follow-up were identical in the 2 groups. CONCLUSION: Although a small, immediate clinical response to IGF-1 injections was seen when combined with training, no additional long-term effect of intratendinous IGF-1 was observed on structural and clinical outcomes in patients with patellar tendinopathy. REGISTRATION: NCT01834989 (ClinicalTrials.gov identifier).

Direct small molecule ADaM-site AMPK activators reveal an AMPKγ3-independent mechanism for blood glucose lowering.

OBJECTIVE: Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK that bind to the allosteric drug and metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. This study aimed to illuminate the mechanisms by which ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e., ADaM-site binding small molecules and the prodrug AICAR. METHODS: The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2 as well as whole-body AMPKγ3-deficient mouse models. In vitro complex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by western blotting in muscle lysate. To investigate the transferability of these studies, we treated diet-induced obese mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle. RESULTS: Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2ß2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2ß2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscles. In vivo PF739 treatment lowered blood glucose levels and increased muscle AMPKγ1-complex activity 2-fold, while AMPKα2ß2γ3 activity was not affected. CONCLUSIONS: ADaM-site binding AMPK activators increase glucose uptake independently of AMPKγ3. Co-incubation with PF739 or 991 and AICAR potentiates the effects on muscle glucose uptake and AMPK activation. In vivo, PF739 lowers blood glucose and selectively activates muscle AMPKγ1-complexes. Collectively, this suggests that pharmacological activation of AMPKγ1-containing complexes in skeletal muscle can increase glucose uptake and can lead to blood glucose lowering.